Heterotandem acyclic peptide complex

Abstract

The present invention relates to a heterotandem acyclic peptide complex comprising a first peptide ligand that binds to a component present on an immune cell, which is conjugated via a linker to a second peptide ligand that binds to a component present on a cancer cell. The present invention also relates to the use of said heterotandem acyclic peptide complex in preventing, inhibiting, or treating cancer.

Description

Technology Field

[0001] The present invention relates to a heterotandem bicyclic peptide complex comprising a first peptide ligand that binds to a component present on an immune cell, which is conjugated to a second peptide ligand that binds to a component present on a cancer cell via a linker. The present invention also relates to the use of said heterotandem bicyclic peptide complex in preventing, inhibiting, or treating cancer. Background Technology

[0002] Cyclic peptides can bind to protein targets with high affinity and target specificity, making them an attractive class of molecules for the development of therapeutics. In fact, numerous cyclic peptides are already being successfully used in clinical practice, examples of which include the antimicrobial peptide vancomycin, the immunosuppressive drug cyclosporine, or the anticancer drug octreotide (Driggers et al . (2008), Nat Rev Drug Discovery 7 (7), 608-24). Good binding properties are generated from the relatively large interaction surface formed between the peptide and the target, as well as from the reduced stereoconformal flexibility of the cyclic structure. Typically, macrocycles bind to a surface of several hundred square angstroms, for example, the cyclic peptide CXCR4 antagonist CVX15 (400 2 ; Wu et al . (2007), Science 330, 1066-71), cyclic peptide having an Arg-Gly-Asp group that binds to integrin αVb3 (355 2 )(Xiong et al . (2002), Science 296 (5565), 151-5) or cyclic peptide inhibitor Eupein-1 (603) that binds to a urokinase-type plasminogen activator 2 ; Zhao et al . (2007), J Struct Biol 160 (1), 1-10) is available.

[0003] Peptide macrocycles are less flexible than linear peptides due to their cyclic arrangement, resulting in less entropy loss and higher binding affinity upon binding to a target. This reduced flexibility also induces the fixation of a target-specific conformation, thereby increasing binding specificity compared to linear peptides. This effect has been exemplified by an efficacious and selective inhibitor of substrate metalloproteinase 8 (MMP-8), which loses its selectivity compared to other MMPs upon ring opening (Cherney et al . (1998), J Med Chem 41 (11), 1749-51). The favorable binding properties achieved through macrocyclization are much more pronounced in multicyclic peptides having more than one peptide ring, such as in vancomycin, nisin, and actinomycin.

[0004] Various research teams have previously grouped polypeptides containing cysteine ​​residues into synthetic molecular structures (Kemp and McNamara (1985), J. Org. Chem; Timmerman et al . (2005), ChemBioChem). Meloen and colleagues used tris(bromomethyl)benzene and related molecules for the rapid and quantitative cyclization of multiple peptide loops on synthetic scaffolds for structural mimicry of protein surfaces (Timmerman et al (2005), ChemBioChem). For example, a method for producing candidate drug compounds by linking a cysteine-containing polypeptide to a molecular scaffold, such as tris(bromomethyl)benzene, is disclosed in WO 2004 / 077062 and WO 2006 / 078161.

[0005] A phage display-based combinatorial approach has been developed to generate and screen a large library of acyclic peptides for targets of interest (Heinis et al . (2009), Nat Chem Biol 5 (7), 502-7 and WO 2009 / 098450). Briefly, a combinatorial library of linear peptides (Cys-(Xaa)6-Cys-(Xaa)6-Cys) containing three cysteine ​​residues and two regions of six random amino acids was displayed on a phage and cyclized by covalently bonding the cysteine ​​side chains to a small molecule (tris-(bromomethyl)benzene).

[0006] According to the first aspect of the present invention,

[0007] (b) a second peptide ligand that binds to a component present on the cancer cell;

[0008] joined via a linker

[0009] (a) Provides a heterotandem acyclic peptide complex comprising a first peptide ligand that binds to a component present on an immune cell, wherein each peptide ligand comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and a molecular scaffold that forms a covalent bond with the cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

[0010] According to a further aspect of the present invention, a pharmaceutical composition is provided comprising a heterotandem acyclic peptide complex as defined herein together with one or more pharmaceutically acceptable excipients.

[0011] According to a further aspect of the present invention, a heterotandem acyclic peptide complex as defined herein is provided for use in preventing, inhibiting, or treating cancer. Brief explanation of the drawing

[0012] Fig. 1: Schematic representation of a heterotandem acyclic peptide complex containing EphA2 and CD137 peptide ligands that bind to both immune cells and cancer cells. Fig. 2: Structure and composition of EphA2-CD137 heterotandem acyclic peptide complex BCY7985. Fig. 3: Analysis of EphA2-CD137 heterotandem acyclic peptide complex BCY7985 in the Promega CD137 luciferase reporter assay (CS196008) in the presence of EphA2-expressing HT1080 cells. Fig. 4: The EphA2 / CD137 heterotandem is active in the CD137 reporter assay, and the activation induction factor depends on the level of tumor target expression in the cell lines used for co-culture. Fig. 5: EphA2 / CD137 heterotandem induces tumor cell death in a primary human T-cell and cancer cell co-culture assay. Tumor cell death is evaluated by counting viable Nucleite Red-positive tumor cells over time. Caspase 3 / 7 dyes are used to identify apoptotic tumor cells. Fig. 6: The nectin-4 / CD137 heterotandem is active in the CD137 reporter assay, and the activation induction factor depends on the tumor target expression levels in the cell lines used for co-culture (HT1376: nectin-4 high and NCI-H292: nectin-4 medium). Fig. 7: The nectin-4 / CD137 heterotandem induces the secretion of IL-2 and IFN-γ cytokines in the PBMC-4T1 co-culture assay. BCY9350 and BCY9351 are non-conjugated controls for nectin-4 and CD137, respectively. Fig. 8:The Nectin-4 / CD137 heterotandem induces target-dependent cytokine release in in vitro cultures of primary patient-derived lung tumors. (A) 3D ellipsoid of in vitro patient-derived tumor cell morphology within 4 h of culture, 10X image under a light microscope. (B) Physiological analysis of Nectin-4 expression in patient-derived tumor samples from three donors. (C) The table shows %CD137 among the three donor samples. + T cells and nectin-4 + (D) Heat map showing the percentage change in immune markers (standardized to vehicle) in response to treatment with control / test compounds. (E) %CD8 in response to treatment with control / test compounds. + ki67 + T cell (vehicle is shown as a dotted line). Fig. 9: The PL-L1 / CD137 heterotandem is active in the CD137 reporter assay in the presence of RKO in PD-L1 expressing cell lines. Fig. 10: Pharmacokinetics of heterotandem in SD rats: BCY10572 and BCY10000 were administered IV at 2 mg / kg (n=3). Specific details for implementing the invention

[0013] According to the first aspect of the present invention,

[0014] (b) Second peptide that binds to a component present on cancer cells

[0015] joined via a linker

[0016] (a) Provides a heterotandem acyclic peptide complex comprising a first peptide ligand that binds to a component present on an immune cell, wherein each peptide ligand comprises a polypeptide comprising at least three cysteine ​​residues separated by at least two loop sequences, and a molecular scaffold that forms a covalent bond with the cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold.

[0017] 1st peptide ligand

[0018] References to the term “immune cell” herein include any cell within the immune system. Suitable examples include leukocytes, e.g., lymphocytes (e.g., T lymphocytes or T cells, B cells or natural killer cells). In one embodiment, the T cell is CD8 or CD4. In a further embodiment, the T cell is CD8. Other examples of immune cells include dendritic cells, follicular dendritic cells, and granulocytes.

[0019] In one embodiment, the component present on the immune cell is CD137.

[0020] CD137 is a member of the Tumor Necrosis Factor (TNF) receptor family. Its other name is Tumor Necrosis Factor Receptor Family Member 9 (TNFRSF9), 4-IBB, and it is induced by lymphocyte activation (ILA). CD137 can be expressed by activated T cells, but to a greater extent on CD8+ T cells than on CD4+ T cells. Additionally, CD137 expression is found on dendritic cells, follicular dendritic cells, natural killer cells, granulocytes, and blood vessel wall cells at the site of inflammation. One characteristic activity of CD137 is its co-stimulatory activity toward activated T cells. Cross-linking of CD137 enhances T cell proliferation, IL-2 secretion, survival, and cytolytic activity. Furthermore, it can enhance immune activity and eliminate tumors in mice.

[0021] CD137 is a T-cell co-stimulatory receptor induced by TCR activation (Nam et al., Curr. Cancer Drug Targets, 5:357-363 (2005); Waits et al., Annu. Rev., Immunol., 23:23-68 (2005)). In addition to expression on activated CD4+ and CD8+ T cells, CD137 is also expressed on CD4+CD25+ regulatory T cells, natural killer (NK) and NK-T cells, monocytes, neutrophils, and dendritic cells. Its natural ligand, CD137L, has been described on antigen-presenting cells including B cells, monocytes / macrophages, and dendritic cells (Watts et al. Annu. Rev., Immunol., 23:23-68 (2005)). When CD137 interacts with its ligand, it leads to increased TCR-induced T-cell proliferation, cytokine production, functional maturation, and prolonged CD8+ T-cell survival (Nam et al, Curr. Cancer Drug Targets, 5:357-363 (2005), Watts et d - l., Annu. Rev. Immunol, 23:23-68 (2005)).

[0022] Signaling via CD137 by activating monoclonal antibodies (mAbs) against CD137L or CD137 results in increased TCR-induced T cell proliferation, cytokine production and functional maturation, and extended CD8+ T cell survival. These effects are generated from (1) activation of the NF-κB, c-Jun NH2-terminal kinase / stress-activated protein kinase (JNK / SAPK), and p38 mitogen-activated protein kinase (MAPK) signaling pathways, and (2) regulation of anti-apoptosis and cell cycle-related gene expression.

[0023] Experiments performed in CD137 and CD127L-deficient mice further demonstrated the importance of CD137 co-stimulation in generating sufficient soluble T cell responses.

[0024] IL-2 and IL-15 activated NK cells express CD137, and ligation of CD137 by agonist mAb stimulates NK cell proliferation and IFN-γ secretion, but does not stimulate cytolytic activity.

[0025] Furthermore, CD137-stimulated NK cells promote the expansion of activated T cells in vitro.

[0026] CD137 agonist mAbs have been shown, depending on their co-stimulatory function, to promote rejection of heart and skin allografts, eradicate established tumors, amplify primary antiviral CD8+ T cell responses, and increase T cell lysis potential. These studies support the view that CD137 signaling promotes T cell function capable of enhancing immunity against tumors and infections.

[0027] In one embodiment, the first peptide ligand comprises a CD137-binding acyclic peptide ligand.

[0028] Suitable examples of CD137-binding acyclic peptide ligands are disclosed in GB patent applications No. 1712589.9 and No. 1802934.8, said peptides are incorporated herein by reference.

[0029] In one embodiment, the CD137-binding acyclic peptide ligand has the following amino acid sequence:

[0030] C i IEEGQYC ii FADPY[Nle]C iii (Sequence No. 1);

[0031] C i [tBuAla]PE[D-Ala]PYC ii FADPY[Nle]C iii (Sequence No. 3);

[0032] C i IEEGQYC ii F[D-Ala]DPY[Nle]C iii (Sequence No. 4);

[0033] C i [tBuAla]PK[D-Ala]PYC ii FADPY[Nle]C iii (Sequence No. 5);

[0034] C i [tBuAla]PE[D-Lys]PYC ii FADPY[Nle]C iii (Sequence No. 6);

[0035] C i [tBuAla]P[K(PYA)][D-Ala]PYC ii FADPY[Nle]C iii (Sequence No. 7);

[0036] C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (Sequence No. 8);

[0037] C i IEE[D-Lys(PYA)]QYC ii FADPY(Nle)C iii (Sequence No. 9); and

[0038] [dC i ][dI][dE][dE][K(PYA)][dQ][dY][dC ii ][dF][dA][dD][dP][dY][dNle][dC iii ] (Sequence No. 10)

[0039] Includes, where C i , C ii and C iii Each represents the first, second, and third cysteine ​​residues, Nle represents norleucine, tBuAla represents t-butyl-alanine, and PYA represents 4-pentinoic acid or its pharmaceutically acceptable salt.

[0040] In one specific embodiment that may be mentioned, the CD137-binding acyclic peptide ligand has the following amino acid sequence:

[0041] C iIEEGQYC ii FADPY[Nle]C iii (Sequence No. 1)

[0042] Includes, where C i , C ii and C iii Each represents a first, second, and third cysteine ​​residue, and Nle represents norleucine, or its pharmaceutically acceptable salt.

[0043] In a further embodiment, the CD137-binding acyclic peptide ligand includes N- and C-terminal modifications.

[0044] Ac-A-(Sequence No. 1)-Dap (hereinafter referred to as BCY7732 in this institution);

[0045] Ac-A-(Sequence No. 1)-Dap(PYA) (hereinafter referred to as BCY7741 in this institution);

[0046] Ac-(Sequence No. 3)-Dap (hereinafter referred to as BCY9172 in this institution);

[0047] Ac-(Sequence No. 3)-Dap(PYA) (hereinafter referred to as BCY11014 in this institution);

[0048] Ac-A-(Sequence No. 4)-Dap (hereinafter referred to as BCY8045 in this institution);

[0049] Ac-(Sequence No. 5)-A (hereinafter referred to as BCY8919 by this institution);

[0050] Ac-(Sequence No. 6)-A (hereinafter referred to as BCY8920 by this institution);

[0051] Ac-(Sequence No. 7)-A (hereinafter referred to as BCY8927 in this institution);

[0052] Ac-(Sequence No. 8)-A (hereinafter referred to as BCY8928 in this institution);

[0053] Ac-A-(Sequence No. 9)-A (hereinafter referred to as BCY7744 in this institution); and

[0054] Ac-[dA]-(Sequence No. 10)-[dA]-NH2 (hereinafter referred to as BCY11506 in this institution)

[0055] It includes, where Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentinoic acid or its pharmaceutically acceptable salt.

[0056] In additional embodiments that may be mentioned, the CD137-binding acyclic peptide ligand comprises N- and C-terminal modifications and amino acid sequence:

[0057] Ac-A-(Sequence No. 1)-Dap (hereinafter referred to as BCY7732 in this institution)

[0058] It includes, where Ac represents an acetyl group and Dap represents diaminopropionic acid, or its pharmaceutically acceptable salt.

[0059] Second peptide ligand

[0060] References to the term "cancer cell" herein include any cell known to be associated with cancer. Cancer cells are generated when genes responsible for regulating cell division are damaged. Carcinogenesis is caused by mutations and upmutations in the genetic material of normal cells, which disrupt the normal balance between proliferation and cell death. This results in uncontrolled cell division and the evolution of said cells through natural selection within the body. The uncontrolled, often rapid, proliferation of said cells can lead to benign or malignant tumors (cancer). Benign tumors do not spread to other parts of the body or invade other tissues. Malignant tumors invade other organs, spread to distant locations (metastasis), and can become life-threatening.

[0061] In one embodiment, the cancer cells are selected from HT1080, SC-OV-3, PC3, H1376, NCI-H292, LnCap, MC38, 4T1-D02 and RKO tumor cells.

[0062] In one embodiment, the component present on the cancer cell is EphA2.

[0063] Eph receptor tyrosine kinase (Eph) belongs to the large group of receptor tyrosine kinases (RTKs), which are kinases that phosphorylate proteins on tyrosine residues. Eph and its membrane-bound ephrine ligand (ephrine) control cell localization and tissue organization (Poliakov et al . (2004) Dev Cell 7, 465-80). Functional and biochemical Eph reactions occur in a higher ligand oligomerization state (Stein et al . (1998) Genes Dev 12, 667-678).

[0064] Various Eph and ephrin have been shown to play a role in vascular development, among other patterning functions. Knockout of EphB4 and ephrin-B2 impairs the ability to remodel the capillary layer into blood vessels (Poliakov et al., cit.) and causes embryonic lethality. Persistent expression of some Eph receptors and ephrin has also been observed in newly formed adult microvessels (Brantley-Sieders et al . (2004) Curr Pharm Des 10, 3431-42; Adams (2003) J Anat 202, 105-12).

[0065] It has been observed that the uncontrolled re-emergence of some ephrines and their receptors in adults also contributes to tumor invasion, metastasis, and neovascularization (Nakamoto et al . (2002) Microsc Res Tech 59, 58-67; Brantley-Sieders et al ., above). Furthermore, it has been found that some members of Eph are overexpressed on tumor cells from various human tumors (Brantley-Sieders et al ., above); Marme (2002) Ann Hematol 81 Suppl 2, S66; Booth et al. (2002) Nat Med 8, 1360-1).

[0066] EPH receptor A2 (ephrin type A receptor 2) is in humans EPHA2 It is a protein encoded by a gene.

[0067] EphA2 is upregulated in many human cancers and is often associated with disease progression, metastasis, and poor prognosis, for example, breast (Zelinski et al (2001) Cancer Res. 61, 2301-2306; Zhuang et al (2010) Cancer Res. 70, 299-308; Brantley-Sieders et al (2011) PLoS One 6, e24426), lung (Brannan et al (2009) Cancer Prev Res (Phila) 2, 1039-1049; Kinch et al (2003) Clin Cancer Res. 9, 613-618; Guo et al (2013) J Thorac Oncol. 8, 301-308), above (Nakamura et al (2005) Cancer Sci. 96, 42-47; Yuan et al (2009) Dig Dis Sci 54, 2410-2417), pancreas (Mudali et al (2006) Clin Exp Metastasis 23, 357-365), Prostate (Walker-Daniels et al (1999) Prostate 41, 275-280), Yang et al (2009) Hepatol Res. 39, 1169-1177) and glioblastoma (Wykosky et al (2005) Mol Cancer Res. 3, 541-551; Li et al (2010) Tumor Biol. 31, 477-488).

[0068] Although the full role of EphA2 in cancer progression has not yet been elucidated, evidence exists for interactions at multiple stages of cancer progression, including tumor cell growth, survival, invasion, and angiogenesis. Downregulation of EphA2 expression inhibits tumor cell spread, whereas (Binda et al (2012) Cancer Cell 22, 765-780), EphA2 blockade inhibits VEGF-induced cell migration (Hess et al (2001) Cancer Res. 61, 3250 - 3255), germination and angiogenesis (Cheng et al (2002) Mol Cancer Res. 1 , 2 - 11 ; Lin et al (2007) Cancer 109, 332-40) and metastatic progression (Brantley-Sieders et al (2005) FASEB J. 19, 1884 - Suppresses 1886).

[0069] Antibody-drug conjugates against EphA2 were shown to significantly reduce tumor growth in rat and mouse xenograft models (Jackson et al (2008) Cancer Research 68, 9367-9374) A similar approach was attempted in humans, but treatment had to be discontinued due to treatment-related side effects (Annunziata et al (2013) Invest New Drugs 31, 77-84).

[0070] In one embodiment, the second peptide ligand comprises an EphA2-binding acyclic peptide ligand.

[0071] Suitable examples of EphA2-binding acyclic peptide ligands are disclosed in GB patent applications No. 1721259.8 and No. 1804102.0, said peptides are incorporated herein by reference.

[0072] In one embodiment, the EphA2-binding acyclic peptide ligand has the following amino acid sequence:

[0073] C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (Sequence No. 2); and

[0074] C i LWDPTPC ii ANLHL[HArg]C iii (Sequence No. 11)

[0075] Includes, where C i , C ii and C iii Each represents a first, second, and third cysteine ​​residue, HyP represents hydroxyproline, dD represents aspartic acid in a D-configuration, and HArg represents homoarginine, or a pharmaceutically acceptable salt thereof.

[0076] In one embodiment that may be mentioned, the EphA2-binding acyclic peptide ligand has the following amino acid sequence:

[0077] C i [HyP]LVNPLC ii LHP[dD]W[HArg]C iii (Sequence No. 2)

[0078] Includes, where C i , C ii and C iii Each represents a first, second, and third cysteine ​​residue, HyP represents hydroxyproline, dD represents aspartic acid in a D-configuration, and HArg represents homoarginine, or its pharmaceutically acceptable salt.

[0079] In a further embodiment, the EphA2-binding acyclic peptide ligand includes an N-terminal modification.

[0080] A-HArg-D-(Sequence No. 2) (hereinafter referred to as BCY9594 by this institution);

[0081] [B-Ala]-[Sar 10 ]-A-[HArg]-D-(Sequence No. 2) (hereinafter referred to as BCY6099 by this institution);

[0082] [PYA]-[B-Ala]-[Sar 10 ]-A-[HArg]-D-(Sequence No. 2) (hereinafter referred to as BCY6169 in this institution); and

[0083] [PYA]-[B-Ala]-[Sar 10 ]-VGP-(Sequence No. 11) (Hereafter referred to as BCY8941)

[0084] Includes, where HArg represents homoarginine, PYA represents 4-pentinoic acid, and Sar 10 represents 10 sarcosine units, and B-Ala represents beta-alanine, or its pharmaceutically acceptable salt.

[0085] In additional embodiments that may be mentioned, the EphA2-binding acyclic peptide ligand includes an N-terminal modification.

[0086] A-HArg-D-(Sequence No. 2) (hereinafter referred to as BCY9594 by this institution)

[0087] It includes, where HArg represents homoarginine, or its pharmaceutically acceptable salt.

[0088] In another embodiment, the component present on the cancer cells is PD-L1.

[0089] Programmed cell death 1 ligand 1 (PD-L1) is a 290-amino acid type 1 transmembrane protein encoded by the CD274 gene on mouse chromosome 19 and human chromosome 9. PD-L1 expression is associated with the evasion of immune responses related to chronic infections, such as chronic viral infections (e.g., HIV, HBV, HCV, and HTLV, among others), chronic bacterial infections (e.g., Helicobacter pylori, among others), and chronic parasitic infections (e.g., Schistosoma mansonii). PD-L1 expression has been detected in numerous tissues and cell types, including T-cells, B-cells, macrophages, dendritic cells, and non-hematopoietic cells including endothelial cells, hepatocytes, and muscle cells, as well as in the placenta.

[0090] PD-L1 expression is also involved in the suppression of anti-tumor immune activity. Tumors express antigens recognizable by host T-cells, but immunological clearance of the tumor is rare. Part of this failure is attributed to immunosuppression by the tumor microenvironment. PD-L1 expression in many tumors is a component of this suppressive environment and acts in cooperation with other immunosuppressive signals. PD-L1 expression has been demonstrated in situ in a wide variety of solid tumors, including breast, lung, colon, ovary, melanoma, bladder, liver, salivary gland, stomach, glioma, thyroid, thymic epithelium, and head and neck (Brown JA et al . 2003 Immunol. 170:1257-66; Dong H et al . 2002 Nat. Med. 8:793-800; Hamanishi J, et al . 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Strome S.E. et al . 2003 Cancer Res. 63:6501-5; Inman B.A. et al . 2007 Cancer 109:1499-505; Konishi J et al. 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al . 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al . 2007 Clin. Cancer Res. 13:2151-57; Thompson R.H. et al . 2004 Proc. Natl. Acad. Sci. USA 101: 17174-79; Wu C et al . 2006 Acta Histochem. 108:19-24). In addition, the expression of the PD-L1 receptor, programmed cell death protein 1 (also known as PD-1 and CD279), is upregulated on tumor-infiltrating lymphocytes, which also contributes to tumor immunosuppression (Blank C et al . 2003 Immunol. 171:4574-81). Most importantly, studies linking oncological PD-L1 expression to disease outcomes demonstrate that PD-L1 expression is strongly correlated with unfavorable prognosis in renal, ovarian, bladder, breast, gastric, and pancreatic cancers (Hamanishi J et al . 2007 Proc. Natl. Acad. Sci. USA 104:3360-65; Inman B.A. et al . 2007 Cancer 109:1499-505; Konishi J et al . 2004 Clin. Cancer Res. 10:5094-100; Nakanishi J et al . 2007 Cancer Immunol. Immunother. 56:1173-82; Nomi T et al . 2007 Clin. Cancer Res. 13:2151-57; Thompson R.H. et al . 2004 Proc. Natl. Acad. Sci. USA 101:17174-79; Wu C et al. 2006 Acta Histochem. 108:19-24). In addition, the above study suggests that higher levels of PD-L1 expression in tumors may promote tumor stage progression and invasion into deeper tissue structures.

[0091] The PD-1 pathway may also play a role in blood cancers. PD-L1 is expressed on many myeloma cells but not on normal plasma cells (Liu J et al . 2007 Blood 110:296-304). PD-L1 is expressed in some primary T-cell lymphomas, particularly anaplastic large cell lymphomas (Brown JA et al , 2003 Immunol. 170:1257-66). PD-1 is highly expressed on T-cells of angioimmunoblastic lymphoma, and PD-L1 is expressed on the associated follicular dendritic cell network (Dorfman DM et al . 2006 Am. J. Surg. Pathol. 30:802-10). In nodular lymphocyte-predominant Hodgkin lymphoma, T-cells associated with lymphocytes or histiocytes (L&H) express PD-1. Microarray analysis using gene readouts induced by PD-1 ligation suggests that tumor-associated T-cells respond to PD-1 signaling in situ in Hodgkin lymphoma (Chemnitz JM et al . 2007 Blood 110:3226-33). PD-1 and PD-L1 are expressed on CD4 T-cells in HTLV-1-mediated adult T-cell leukemia and lymphoma (Shimauchi T et al . 2007 Int. J. Cancer 121: 2585-90). These tumor cells are low-responsive to TCR signaling.

[0092] Studies in animal models demonstrate that tumor-associated PD-L1 inhibits T-cell activation and tumor cell lysis, and in some cases induces increased tumor-specific T-cell death (Dong H et al. 2002 Nat. Med. 8:793-800; Hirano F et al . 2005 Cancer Res. 65:1089-96). Tumor-associated APCs can also utilize the PD-1:PD-L1 pathway to regulate anti-tumor T-cell responses. PD-L1 expression in tumor-associated myeloid DC populations is upregulated by tumor environmental factors (Curiel TJ et al . 2003 Nat. Med. 9:562-67). Plasmacytoid dendritic cells (DCs) in tumor-draining lymph nodes of B16 melanoma express IDO, which strongly activates the inhibitory activity of regulatory T-cells. The inhibitory activity of IDO-treated regulatory T-cells required cell contact with IDO-expressing DCs (Sharma MD et al . 2007 Clin. Invest. 117:2570-82).

[0093] In one embodiment, the second peptide ligand comprises a PD-L1 binding acyclic peptide ligand.

[0094] Suitable examples of PD-L1 binding acyclic peptide ligands are disclosed in GB patent applications No. 1820956.9 and No. 1820969.2, said peptides are incorporated herein by reference.

[0095] In one embodiment, the PD-L1 binding acyclic peptide ligand is

[0096] C i [HArg]DWC ii HWTFSHGHPC iii (Sequence No. 12);

[0097] C i SAGWLTMC ii QKLHLC iii (Sequence No. 13); and

[0098] C i SAGWLTMC ii Q[K(PYA)]LHLC iii (Sequence No. 14)

[0099] It includes an amino acid sequence selected from among, wherein C i , C ii and C iii Each represents the first, second, and third cysteine ​​residues, HArg represents homoarginine, and PYA represents 4-pentinoic acid, or its pharmaceutically acceptable salt.

[0100] In a further embodiment, the PD-L1 binding acyclic peptide ligand comprises N-terminal and / or C-terminal modifications.

[0101] [PYA]-[B-Ala]-[Sar 10 ]-(Sequence No. 12) (hereinafter referred to as BCY8938 by this institution);

[0102] [PYA]-[B-Ala]-[Sar 10 ]-SDK-(Sequence No. 13) (hereinafter referred to as BCY10043 by this institution);

[0103] NH2-SDK-(Sequence No. 13)-[Sar 10 ]-[K(PYA)] (hereinafter referred to as BCY10044 by this institution);

[0104] NH2-SDK-(Sequence No. 14) (hereinafter referred to as BCY10045 in this institution); and

[0105] Ac-SDK-(Sequence No. 14)-PSH (hereinafter referred to as BCY10861 in this institution)

[0106] Includes, where PYA represents 4-pentinoic acid, B-Ala represents beta-alanine, and Sar 10 It represents 10 sarcosine units, or its pharmaceutically acceptable salt.

[0107] In another embodiment, the component present on the cancer cell is nectin-4.

[0108] Nectin-4 is a surface molecule belonging to the nectin family, a group of proteins comprising four members. Nectins are cell adhesion molecules that play a role in various biological processes, such as polarity, proliferation, differentiation, and migration, in epithelial, endothelial, immune, and neuronal cells during development and adulthood. They are involved in numerous pathological processes in humans. They are major receptors for poliovirus, herpes simplex virus, and measles virus. Mutations in the genes encoding Nectin-1 (PVRL1) or Nectin-4 (PVRL4) cause ectodermal dysplasia syndromes associated with other abnormalities. Nectin-4 is expressed during fetal development. In adult tissues, its expression is more restricted than that of other members of the family. Nectin-4 is a tumor-associated antigen in 50%, 49%, and 86% of breast, ovarian, and lung carcinomas, respectively, which are typically associated with a poor prognosis. Its expression is not detected in the corresponding normal tissues. In breast tumors, nectin-4 is primarily expressed in triple-negative and ERBB2+ carcinomas. In the serum of patients with these cancers, the detection of the soluble form of nectin-4 is associated with a poor prognosis. Serum nectin-4 levels increase during metastatic progression and decrease after treatment. These results suggest that nectin-4 may be a reliable target for cancer treatment. Accordingly, a number of anti-nectin-4 antibodies have been described in the prior art. In particular, enfortumab vedotin (ASG-22ME) is an antibody-drug conjugate (ADC) that targets nectin-4 and is currently being clinically studied for the treatment of patients with solid tumors.

[0109] In one embodiment, the second peptide ligand comprises a nectin-4 binding acyclic peptide ligand.

[0110] Suitable examples of nectin-4 binding acyclic peptide ligands are disclosed in GB patent applications No. 1810250.9, No. 1815684.4 and No. 1818499.4, said peptides are incorporated herein by reference.

[0111] In one embodiment, the nectin-4 binding acyclic peptide ligand is

[0112] C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (Sequence No. 15; hereinafter referred to herein as BCY8116);

[0113] C i P[1Nal][dD]C ii M[HArg]D[dW]STP[HyP][dW]C iii (Sequence No. 16; hereinafter referred to herein as BCY11415); and

[0114] C i P[1Nal][dK](Sar 10 -(B-Ala))C ii M[HArg]DWSTP[HyP]WC iii (Sequence No. 17);

[0115] C i PFGC ii M[HArg]DWSTP[HyP]WC iii (Sequence No. 18; hereinafter referred to herein as BCY11414)

[0116] It includes an amino acid sequence selected from among, wherein C i , C ii and C iii represents the first, second, and third cysteine ​​residues, respectively, 1Nal represents 1-naphthylalanine, HArg represents homoarginine, HyP represents hydroxyproline, and Sar 10 represents 10 sarcosine units, and B-Ala represents beta-alanine, or its pharmaceutically acceptable salt.

[0117] In a further embodiment, the nectin-4 binding acyclic peptide ligand includes an N-terminal modification.

[0118] Sequence No. 15 (hereinafter referred to as BCY8116 by this institution);

[0119] [PYA]-[B-Ala]-[Sar 10 ]-(Sequence No. 15) (hereinafter referred to as BCY8846 by this institution);

[0120] Sequence No. 16 (hereinafter referred to as BCY11415 by this institution);

[0121] [PYA]-[B-Ala]-[Sar 10 ]-(Sequence No. 16) (hereinafter referred to as BCY11942 by this institution);

[0122] Ac-(Sequence No. 17) (hereinafter referred to as BCY8831 in this institution); and

[0123] Sequence No. 18 (hereinafter referred to as BCY11414 by this institution)

[0124] Includes, where PYA represents 4-pentinoic acid, B-Ala represents beta-alanine, and Sar 10 It represents 10 sarcosine units, or its pharmaceutically acceptable salt.

[0125] In another embodiment, the component present on the cancer cells is prostate-specific membrane antigen (PSMA).

[0126] Prostate-specific membrane antigen (PSMA) (also known as glutamate carboxypeptidase II (GCPII), N-acetyl-L-aspartyl-L-glutamate peptidase I (NAALADase I), and NAAG peptidase) in humans FOLH1 It is an enzyme encoded by the (folate hydrolase 1) gene. Human GCPII contains 750 amino acids and weighs approximately 84 kDa.

[0127] Human PSMA is highly expressed in the prostate, approximately 100 times more than in most other tissues. In some prostate cancers, PSMA is the second most upregulated gene product, increasing 8 to 12-fold compared to levels in non-cancerous prostate cells. Due to this high expression, PSMA is being developed as a potential biomarker for the treatment and imaging of some cancers. In human prostate cancer, tumors with higher expression are associated with a faster time to progression and a higher percentage of patients with recurrence.

[0128] In one embodiment, the second peptide ligand comprises a PSMA-bound acyclic peptide ligand.

[0129] Suitable examples of PSMA-bound acyclic peptide ligands are disclosed in GB patent applications No. 1810318.4, No. 1810325.9 and No. 1820325.7, said peptides are incorporated herein by reference.

[0130] Linker

[0131] It will be known that a first peptide ligand can be conjugated to a second peptide ligand via any suitable linker. Typically, the design of the linker will be such that the two acyclic peptides bind to their respective targets individually without interference, or bind to two target receptors simultaneously. Additionally, the linker must allow simultaneous binding to two targets while maintaining a suitable distance between target cells to produce the desired functional result. The properties of the linker can be adjusted to increase length, stiffness, or solubility to optimize the desired functional result. The linker can also be designed to allow the attachment of more than one acyclic to the same target. An increase in the binding valence of any one of the conjugated peptides may act to increase the affinity of the heterotandem to the target cells or contribute to inducing the oligomerization of one or both of the target receptors.

[0132] In one embodiment, the linker is selected from the following sequences: -CH2-, -PEG5-, -PEG 10 -, -PEG 12 -, -PEG 23 -, -PEG 24 -, -PEG 15 -Sar5-, -PEG 10 -Sar 10 -, -PEG5-Sar 15 -, -PEG5-Sar5-, -B-Ala-Sar 20 -, -B-Ala-Sar 10 -PEG 10 -, -B-Ala-Sar5-PEG 15 - and -B-Ala-Sar5-PEG5-.

[0133] The structural representation of the appropriate linker is shown in detail:

[0134]

[0135] Heterotandem complex

[0136] In one specific embodiment, the first peptide ligand comprises a CD137-binding acyclic peptide ligand attached to a TATA scaffold, and the second peptide ligand comprises an EphA2-binding acyclic peptide ligand attached to a TATA scaffold, and the heterotandem complex is selected from the following:

[0137]

[0138] Heterotandem acyclic peptide complex BCY7985 is PEG 12 It consists of CD137-specific peptide BCY7859 connected to the N-terminal PYA group of EphA2-specific peptide BCY6169 (illustrated in Fig. 2).

[0139] CD137 is a homotrimeric protein, and the natural ligand CD137L exists as an expressed or secreted homotrimer on immune cells. The biology of CD137 is highly dependent on multimerization to induce CD137 activity in immune cells. One way to generate CD137 multimerization is through cellular cross-linking of CD137-specific agonists via interaction with specific receptors present on other cells.

[0140] EphA2 is highly expressed on tumor cells, and the oligomerization of the receptor tyrosine kinase by the ephrin-A ligand drives its activation. Without being bound by theory, the inventors hypothesize that an EphA2-CD137 heterotandem, consisting of one EphA2-specific peptide bound to one CD137-specific peptide, acts to cross-link CD137. The implication is that CD137 will be multimeric and activated in the presence of EphA2 on cells such as tumor cells. This will drive CD137 immune cell activation in the local tumor environment (Fig. 1).

[0141] This hypothesis was tested in the CD137 cell activity reporter assay described herein, and the results are shown in FIG. 3 of this specification, where it can be seen that BCY7985 showed strong induction of CD137 cell activity in the Promega CD137 luciferase reporter assay (CS196008) in the presence of EphA2-expressing HT1080 cells.

[0142] In one other specific embodiment, the first peptide ligand comprises a CD137-binding acyclic peptide ligand attached to a TATA scaffold, and the second peptide ligand comprises a nectin-4-binding acyclic peptide ligand attached to a TATA scaffold, and the heterotandem complex is selected from the following:

[0143]

[0144] Although we do not wish to be bound by theory, the inventors believe that a nectin-4-CD137 heterotandem consisting of one nectin-4-specific peptide coupled to one CD137-specific peptide acts to cross-link CD137 in the same manner as previously described herein for EphA2.

[0145] In one embodiment, the nectin-4-CD137 heterotandem is other than any one or more of BCY11857, BCY11858 and / or BCY11859.

[0146] In one other specific embodiment, the first peptide ligand comprises a CD137-binding acyclic peptide ligand attached to a TATA scaffold, and the second peptide ligand comprises a PD-L1-binding acyclic peptide ligand attached to a TATA scaffold, and the heterotandem complex is selected from the following:

[0147]

[0148] Although we do not wish to be bound by theory, the inventors believe that a PD-L1-CD137 heterotandem consisting of one PD-L1-specific peptide coupled to one CD137-specific peptide acts to cross-link CD137 in the same manner as previously described herein for EphA2.

[0149] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art, e.g., peptide chemistry, cell culture and phage display, nucleic acid chemistry, and biochemistry. Standard techniques are used in molecular biology, genetics, and biochemical methods (see the following references cited herein by reference: Sambrook et al ., Molecular Cloning: A Laboratory Manual, 3rd ed., 2001, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel et al ., Short Protocols in Molecular Biology (1999) 4 th ed., John Wiley & Sons, Inc.).

[0150] nomenclature

[0151] Numbering

[0152] When referring to the position of an amino acid residue within the compound of the present invention, the cysteine ​​residue (C i , C ii and C iii Since ) does not change, it is omitted from the numbering; therefore, the numbering of amino acid residues in Sequence No. 1 is referred to as follows:

[0153] C i -I1-E2-E3-G4-Q5-Y6-C ii -F7-A8-D9-P 10 -Y 11 -[Nle]12 -C iii (Sequence No. 1)

[0154] For the purposes of the foregoing, all acyclic peptides are presumed to be cyclic with TBMB (1,3,5-tris(bromomethyl)benzene) or 1,1',1"-(1,3,5-triazinan-1,3,5-triyl)triprop-2-en-1-one (TATA) to produce a 3-substituted structure. Cyclication by TBMB and TATA is C i , C ii and C iii It happens on the surface.

[0155] molecular format

[0156] An N- or C-terminal extension into the acyclic core sequence is added to the left or right of the sequence, separated by a hyphen. For example, the N-terminal βAla-Sar10-Ala tail will be represented as follows:

[0157] βAla-Sar10-A-(Sequence No. X).

[0158] Inverse peptide sequence

[0159] Literature [Nair et al In light of the disclosure of

[2003] J Immunol 170(3), 1362-1373], it is expected that the peptide sequence disclosed herein may also be used in its retro-inverse form. For example, said sequence is inverted (i.e., the N-terminus becomes the C-terminus and vice versa), and its stereochemistry is likewise inverted (i.e., the D-amino acid becomes the L-amino acid and vice versa).

[0160] peptide ligand

[0161] A peptide ligand as referred to herein refers to a peptide covalently bonded to a molecular scaffold. Typically, such a peptide comprises two or more reactive groups (i.e., cysteine ​​residues) capable of forming a covalent bond to said scaffold, and a sequence opposite between said reactive groups, referred to as a loop sequence, so that said peptide forms a loop when bound to said scaffold. In the case of the present invention, said peptide comprises at least three cysteine ​​residues (C in this invention i , C ii and C iii It includes (referred to as) and forms at least two loops on the scaffold.

[0162] Pharmaceutically acceptable salts

[0163] It will be understood that the salt form is within the scope of the present invention, and that references to peptide ligands include the salt form of said ligand.

[0164] The salt of the present invention is obtained by conventional chemical methods, e.g., literature [ Pharmaceutical Salts: Properties, Selection, and Use A salt containing a basic or acidic portion can be synthesized from a parent compound containing a basic or acidic portion by the method described in [ , P. Heinrich Stahl (Editor), Camille G. Wermuth (Editor), ISBN: 3-90639-026-8, Hardcover, 388 pages, August 2002]. Generally, such a salt can be prepared by reacting the free acid or basic form of the compound with a suitable base or acid in an aqueous or organic solvent, or a mixture of the two.

[0165] Acid addition salts (mono- or di-salts) can be formed from a wide variety of acids (both inorganic and organic). Examples of acid addition salts are acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid (e.g., L-ascorbic acid), L-aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, butanoic acid, (+) camphoric acid, camphor-sulfonic acid, (+)-(1S)-camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, cinnamic acid, citric acid, cyclamic acid, dodecyl sulfate, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, D-gluconic acid, glucuronic acid (e.g., D-glucuronic acid), glutamic acid (e.g., L-glutamic acid), α-oxoglutaric acid, Glycolic acid, hipfuric acid, hydroxylases (e.g., hydrobromide, hydrochloric acid, hydroiodide), isethionic acid, lactic acid (e.g., (+)-L-lactic acid, (±)-DL-lactic acid), lactobionic acid, maleic acid, malic acid, (-)-L-malic acid, malonic acid, (±)-DL-mandelic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, nitric acid, oleic acid, orotic acid, oxalic acid, palmitic acid, parmoic acid, phosphoric acid, propionic acid, pyruvate, L-pyroglutamic acid, salicylic acid, 4-amino-salicylic acid, sevacic acid, stearic acid, succinic acid, sulfuric acid, tannic acid, (+)-L-tartaric acid, It includes a one- or two-salt formed with an acid selected from the group consisting of thiocyanate, p-toluenesulfonic acid, undecylenic acid, and valeric acid, as well as acylated amino acids and cation exchange resins.

[0166] One specific group of salts consists of salts formed from acetic acid, hydrochloric acid, hydroiodic acid, phosphoric acid, nitric acid, sulfuric acid, citric acid, lactic acid, succinic acid, maleic acid, malic acid, isethionic acid, fumaric acid, benzenesulfonic acid, toluenesulfonic acid, sulfuric acid, methanesulfonic acid (mesylate), ethanesulfonic acid, naphthalenesulfonic acid, valeric acid, propanoic acid, butanoic acid, malonic acid, glucuronic acid, and lactobionic acid. One specific salt is a hydrochloride salt. Another specific salt is an acetate salt.

[0167] When the compound is anionic or has a functional group that can be anionic (e.g., -COOH is -COO - (It may be), salts can be formed with organic or inorganic bases to produce suitable cations. Examples of suitable inorganic cations are, but are not limited to, alkali metal ions, e.g., Li + , Na + and K + , alkaline earth metal cations, e.g., Ca 2+ and Mg 2+ , and other cations, e.g., Al 3+ or Zn + Includes. Examples of suitable organic cations are, without limitation, ammonium ions (i.e., NH4). + ) and substituted ammonium ions (e.g., NH3R + , NH2R2 + , NHR3 + , NR4 + Includes ). Some suitable substituted ammonium ions are those derived from amino acids, e.g., lysine and arginine, as well as methylamine, ethylamine, diethylamine, propylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine, and tromethamine. Common examples of quaternary ammonium ions are N(CH3)4 + am.

[0168] When a compound of the present invention contains an amine functional group, it may form a quaternary ammonium salt by reacting with an alkylating agent according to a method known to those skilled in the art, for example. Such a quaternary ammonium compound is within the scope of the present invention.

[0169] modified derivative

[0170] It will be understood that modified derivatives of peptide ligands as defined herein are within the scope of the present invention. Examples of such suitable modified derivatives include N-terminal and / or C-terminal modifications; replacement of one or more amino acid residues with one or more non-natural amino acid residues (e.g., replacement of one or more polar amino acid residues with one or more isoelectronic amino acids; replacement of one or more non-polar amino acid residues with other non-natural isoelectronic amino acids); addition of a dilatation group; replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues; replacement of one or more amino acid residues with alanine; replacement of one or more L-amino acid residues with one or more D-amino acid residues; N-alkylation of one or more amide bonds within a cyclic peptide ligand; replacement of one or more peptide bonds with substitute bonds; modification of the peptide main chain length; It includes one or more modifications selected from substitution of an alpha-carbon hydrogen of one or more amino acid residues by another chemical group, modification of said amino acids by suitable amine, thiol, carboxylic acid, and phenol-reactive reagents for the functionalization of said amino acids such as cysteine, lysine, glutamate / aspartate, and tyrosine, and introduction or replacement of an azide or alkyne-containing amino acid that allows functionalization by an amino acid that introduces orthogonal reactivity suitable for functionalization, for example, an alkyne or azide-containing moiety, each of which allows functionalization by an azide or alkyne-containing amino acid.

[0171] In one embodiment, the modified derivative comprises N-terminal and / or C-terminal modifications. In a further embodiment, the modified derivative comprises an N-terminal modification using a suitable amino-reactive chemistry, and / or a C-terminal modification using a suitable carboxy-reactive chemistry. In a further embodiment, the N-terminal or C-terminal modification comprises the addition of an effector, e.g., a cytotoxic agent, a radiochelating agent, or a chromophore, but not limited to.

[0172] In a further embodiment, the modified derivative comprises an N-terminal modification. In a further embodiment, the N-terminal modification comprises an N-terminal acetyl group. In the above embodiment, an N-terminal cysteine ​​group (C in the present invention) i The group referred to as () is capped with acetic anhydride or other suitable reagent during peptide synthesis to induce an N-terminal acetylated molecule. The above embodiment provides the advantage of removing potential recognition sites for aminopeptidase and eliminates the possibility of degradation of acyclic peptides.

[0173] In another embodiment, the N-terminal modification includes the addition of a molecular spacer group that promotes the conjugation of an effector group and the maintenance of the acyclic peptide's efficacy toward its target.

[0174] In a further embodiment, the modified derivative comprises a C-terminal modification. In a further embodiment, the C-terminal modification comprises an amide group. In the above embodiment, the C-terminal cysteine ​​group (C in the present invention) iii A group referred to as (as) is synthesized as an amide during peptide synthesis to induce a C-terminal amidated molecule. The above embodiment provides the advantage of removing potential recognition sites for carboxypeptidase and reduces the proteolytic degradation potential of acyclic peptides.

[0175] In one embodiment, the modified derivative comprises the replacement of one or more amino acid residues with one or more non-natural amino acid residues. In the above embodiment, a non-natural amino acid having an isoelectronic side chain that is not recognized by degradable proteases and does not have any adverse effect on target efficacy may be selected.

[0176] On the other hand, non-natural amino acids having restricted amino acid side chains may be used so that proteolytic hydrolysis of nearby peptide bonds is stereochemically and stereochemically inhibited. In particular, the above is a proline analog, bulky side chain, C α - It relates to disubstituted derivatives (e.g., aminoisobutyric acid, Aib), and simple derivatives such as cycloamino acids and amino-cyclopropylcarboxylic acids.

[0177] In one embodiment, the modified derivative comprises the addition of a dilatation group. In a further embodiment, the modified derivative comprises N-terminal cysteine ​​(C i ) and / or C-terminal cysteine ​​(C iii Includes the addition of this spacing group to ).

[0178] In one embodiment, the modified derivative comprises the replacement of one or more oxidation-sensitive amino acid residues with one or more oxidation-resistant amino acid residues. In a further embodiment, the modified derivative comprises the replacement of a tryptophan residue with a naphthylalanine or alanine residue. The embodiment provides the advantage of improving the pharmaceutical stability profile of the resulting acyclic peptide ligand.

[0179] In one embodiment, the modified derivative comprises the replacement of one or more charged amino acid residues with one or more hydrophobic amino acid residues. In another embodiment, the modified derivative comprises the replacement of one or more hydrophobic amino acid residues with one or more charged amino acid residues. The precise balance of charged to hydrophobic amino acid residues is an important feature of the acyclic peptide ligand. For example, hydrophobic amino acid residues affect plasma protein binding and, thus, the concentration of the available free fraction in plasma, while charged amino acid residues (particularly arginine) may affect the interaction between the peptide and the phospholipid membrane on the cell surface. Together, these two can affect the half-life, distribution volume, and exposure of the peptide drug, which can be tailored to clinical endpoints. Additionally, the precise combination and number of charged to hydrophobic amino acid residues can reduce irritation at the injection site (when the peptide drug is administered subcutaneously).

[0180] In one embodiment, the modified derivative comprises the replacement of one or more L-amino acid residues with one or more D-amino acid residues. This embodiment is believed to increase proteolytic stability by steric hindrance and a tendency to stabilize the β-turn conformation of the D-amino acid (Tugyi et al (2005) PNAS, 102(2), 413-418).

[0181] In one embodiment, the modified derivative comprises the removal of any amino acid residue and substitution with alanine. The embodiment provides the advantage of removing potential proteolytic attack site(s).

[0182] It should be noted that each of the aforementioned modifications acts to intentionally improve the efficacy or stability of the peptide. Further efficacy improvements based on modifications can be achieved through the following mechanisms:

[0183] - Integration of hydrophobic parts that utilize hydrophobic effects and induce a lower off-rate, thereby achieving higher affinity;

[0184] - Integration of charged groups utilizing long-range ionic interactions to induce a faster binding rate (on rate) and higher affinity (e.g., literature [Schreiber et al , Rapid, electrostatically assisted association of proteins See (1996), Nature Struct. Biol. 3, 427-31); and

[0185] - For example, additional restrictions are incorporated within the peptide by precisely restricting the side chains of amino acids to minimize entropy loss upon target binding, restricting the twist angle of the main chain to minimize entropy loss upon target binding, and introducing additional cyclization into the molecule for the same reason.

[0186] (For the review, literature [Gentilucci et al , Curr. Pharmaceutical Design, (2010), 16, 3185-203], and literature [Nestor et al See Curr. Medicinal Chem (2009), 16, 4399-418).

[0187] Isotope change

[0188] The present invention comprises all pharmaceutically acceptable (radioactive)isotope-labeled peptide ligands of the present invention in which one or more atoms are replaced by atoms having the same number of atoms but different atomic mass or mass number from that which is ordinarily found in nature, and peptide ligands of the present invention (referred to as “effect groups”) attached to retain the associated (radioactive)isotope, and peptide ligands of the present invention in which some functional groups are covalently replaced by the associated (radioactive)isotope or isotope-labeled functional groups.

[0189] Examples of isotopes suitable for inclusion in the peptide ligand of the present invention include hydrogen, for example. 2 H(D) and 3 H(T), carbon, for example 11 C, 13 C and 14 C, chlorine, for example 36 Cl, fluorine, for example 18 F, iodine, for example 123 I, 125 I and 131 I, nitrogen, for example, 13 N and 15 N, oxygen, for example 15 O, 17 O and 18 O, person, for example 32 P, yellow, for example 35 S, copper, for example 64 Cu, gallium, for example 67 Ga or 68 Ga, yttrium, for example 90 Y and lutetium, for example 177 Lu, and bismuth, for example 213 It includes isotopes of Bi.

[0190] Some of the isotope-labeled peptide ligands of the present invention, for example those containing radioisotopes, are useful for studying drug and / or substrate tissue distribution and for clinically evaluating the presence and / or absence of nectin-4 targets in diseased tissues. The peptide ligands of the present invention may also possess valuable diagnostic properties in that they can be used to detect or identify the formation of complexes between the labeled compound and other molecules, peptides, proteins, enzymes, or receptors. The detection or identification method may use a compound labeled with a labeling agent, for example, a radioisotope, enzyme, fluorescent material, luminescent material (e.g., luminol, luminol derivatives, luciferin, aquorin, and luciferase), etc. The radioisotope tritium, i.e. 3 H(T) and carbon-14, i.e. 14C is particularly useful for the above purpose in light of its ease of integration and immediate detection means.

[0191] Heavier isotopes, for example, deuterium, that is 2 Substitution by H(D) can provide some therapeutic advantages resulting from greater metabolic stability, e.g., increased in vivo half-life or reduced dosage requirements, and thus may be desirable in some situations.

[0192] Positron-emitting isotopes, for example 11 C, 18 F, 15 O and 13 Substitution by N can be useful for positron emission tomography (PET) studies to examine target occupancy.

[0193] The isotope-labeled compound of the peptide ligand of the present invention can be prepared by conventional techniques generally known to those skilled in the art, or by a process similar to that described in the attached examples using a suitable isotope-labeled reagent instead of the previously used unlabeled reagent.

[0194] molecular scaffold

[0195] Molecular scaffolds are described, for example, in WO 2009 / 098450 and the references cited therein, in particular WO 2004 / 077062 and WO 2006 / 078161.

[0196] As recorded in the above document, the molecular scaffold can be a small molecule, for example, a small organic molecule.

[0197] In one embodiment, the molecular scaffold may be a macromolecule. In one embodiment, the molecular scaffold is a macromolecule composed of amino acids, nucleotides, or carbohydrates.

[0198] In one embodiment, the molecular scaffold includes a reactive group capable of reacting with the functional group(s) of the polypeptide to form a covalent bond.

[0199] The molecular scaffold may include chemical groups that form a bond with the peptide, for example, amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, azides, anhydrides, succinimide, maleimide, alkyl halides, and acyl halides.

[0200] In one embodiment, the molecular scaffold may comprise or be composed of hexahydro-1,3,5-triazine, particularly 1,3,5-triacryloylhexahydro-1,3,5-triazine ('TATA'), or derivatives thereof.

[0201] In one embodiment, the molecular scaffold is 2,4,6-tris(bromomethyl)methylene. The molecule is similar to 1,3,5-tris(bromomethyl)benzene (TBMB) but contains three additional methyl groups attached to the benzene ring. This has the advantage that the additional methyl groups can form additional contacts with the polypeptide and thus add additional structural constraints.

[0202] The molecular scaffold of the present invention contains a chemical group that enables a functional group of a polypeptide of the encoded library of the present invention to form a covalent bond with said molecular scaffold. The chemical group is selected from a wide variety of functional groups including amines, thiols, alcohols, ketones, aldehydes, nitriles, carboxylic acids, esters, alkenes, alkynes, anhydrides, succinimide, maleimide, azide, alkyl halides, and acyl halides.

[0203] The scaffold reactive group that can be used on the molecular scaffold for reaction with the thiol group of cysteine ​​is an alkyl halide (or is also named a halogenalkane or haloalkane).

[0204] Examples include bromomethylbenzene (a scaffold reactor exemplified by TBMB) or iodoacetamide. Other scaffold reactors used to selectively bind compounds to cysteine ​​in proteins are maleimides, αβ-unsaturated carbonyl-containing compounds, and α-halomethylcarbonyl-containing compounds. Examples of maleimides that can be used as molecular scaffolds in the present invention include tris-(2-maleimidoethyl)amine, tris-(2-maleimidoethyl)benzene, and tris-(maleimido)benzene. An example of an αβ-unsaturated carbonyl-containing compound is 1,1',1"-(1,3,5-triazinan-1,3,5-triyl)triprop-2-en-1-one (TATA) (Angewandte Chemie, International Edition (2014), 53(6), 1602-1606). An example of an α-halomethylcarbonyl-containing compound is N,N',N"-(benzene-1,3,5-triyl)tris(2-bromoacetamide). Selenocysteine ​​is also a natural amino acid with reactivity similar to cysteine ​​and can be used in the same reaction. Therefore, whenever cysteine ​​is mentioned, selenocysteine ​​is typically allowed as a substitute unless otherwise indicated in the context.

[0205] synthesis

[0206] The peptide of the present invention can be synthetically prepared by standard techniques and then reacted with a molecular scaffold in vitro. When performing this, standard chemistry may be used. This enables the rapid, large-scale production of soluble materials for further downstream experiments or verification. The above method is described using conventional chemistry, for example, the literature [Timmerman et al It could be performed using what was disclosed in ](above).

[0207] Accordingly, the present invention also relates to the preparation of a selected polypeptide or conjugate as set forth herein, wherein said preparation comprises any additional steps as described below. In one embodiment, these steps are performed on a final product polypeptide / conjugate prepared by chemical synthesis.

[0208] Amino acid residues in the polypeptide of interest may be optionally substituted when preparing a conjugate or complex.

[0209] Peptides can be extended to incorporate, for example, another loop and thus introduce multiple specificities.

[0210] To extend the peptide, the above can be chemically extended simply at its N-terminus or C-terminus or within the loop using orthogonally protected lysine (and analogs) using standard solid-phase or solution-phase chemistry. An activated or activable N- or C-terminus can be introduced using standard (biological)conjugation techniques. Alternatively, addition, e.g., [Dawson] et al As described in . 1994. Synthesis of Proteins by Native Chemical Ligation. Science 266:776-779], by fragment condensation or native chemical ligation, or by enzymes, for example, in the literature [Chang et al. Proc Natl Acad Sci US A. 1994 Dec 20; 91(26):12544-8] or literature[Hikari et al As described in Bioorganic & Medicinal Chemistry Letters Volume 18, Issue 22, 15 November 2008, Pages 6000-6003, this can be performed using a subtiligase.

[0211] On the other hand, the peptide can be extended or modified by additional conjugation via a disulfide bond. This has the additional advantage that the first and second peptides are separated from each other once they are in the reducing environment of the cell. In this case, a molecular scaffold (e.g., TBMB) could be added during the chemical synthesis of the first peptide for reaction with three cysteine ​​groups; subsequently, additional cysteine ​​or thiol could be attached to the N or C-terminus of the first peptide, so that the cysteine ​​or thiol reacts only with the free cysteine ​​or thiol of the second peptide to form a disulfide-linked acyclic peptide-peptide conjugate.

[0212] A similar technique is applied equally to the synthesis / combination of two acyclic and bispecific macrocycles to potentially produce a quadruplicative molecule.

[0213] Furthermore, the addition of other functional or effector groups can be carried out in the same manner using suitable chemistry at the N- or C-terminus or via the side chain. In one embodiment, the bonding is carried out in such a manner that it does not block the activity of any of the entities.

[0214] Pharmaceutical composition

[0215] According to a further aspect of the present invention, a pharmaceutical composition is provided comprising a peptide ligand as defined herein together with one or more pharmaceutically acceptable excipients.

[0216] Generally, the peptide ligand of the present invention may be used in a purified form with a pharmacologically suitable excipient or carrier. Typically, these excipients or carriers comprise aqueous or alcohol / aqueous solutions, emulsions, or suspensions containing saline and / or buffered media. Parenteral vehicles comprise sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride and lactated Ringer's solution. Suitable physiologically acceptable adjuvants may be selected from thickeners, e.g., carboxymethylcellulose, polyvinylpyrrolidone, gelatin, and alginate, if necessary to maintain the polypeptide complex in the suspension.

[0217] Intravenous vehicles include fluids, nutritional supplements, and electrolyte supplements, e.g., Ringer's dextrose-based supplements. Preservatives and other additives, e.g., antimicrobial agents, antioxidants, chelating agents, and inert gases may also be present (Mack (1982) Remington's Pharmaceutical Sciences, 16th Edition).

[0218] The peptide ligand of the present invention may be used as a composition administered separately or in combination with other agents. The above may include antibodies, antibody fragments and various immunotherapeutic drugs, e.g., cyclosporine, methotrexate, adriamycin or cisplatinium, and immunotoxins. The pharmaceutical composition may include a “cocktail” of a combination of the protein ligand of the present invention and a selected polypeptide according to the present invention having different specificities, e.g., a combination of selected polypeptides using different target ligands, with various cytotoxic or other agents, regardless of whether they are combined prior to administration.

[0219] The route of administration of the pharmaceutical composition according to the present invention may be any route commonly known to those skilled in the art. For therapeutic purposes, the peptide ligand of the present invention may be administered to any patient according to standard techniques. Such administration may be carried out by any suitable method, for example, parenterally, intravenously, intramuscularly, intraperitoneally, percutaneously, or pulmonaryly, or suitably by direct infusion via a catheter. Preferably, the pharmaceutical composition according to the present invention will be administered by inhalation. The dosage and frequency of administration will vary depending on the patient's age, sex and condition, concomitant use of other drugs, contraindications, and other parameters that the physician must consider.

[0220] The peptide ligand of the present invention may be freeze-dried for storage and reconstituted in a suitable carrier before use. The above technique has been proven to be effective and is a freeze-drying method known in the art, and a reconstitution technique may be used. Those skilled in the art will know that freeze-drying and reconstitution can result in varying degrees of loss of activity and may need to be adjusted toward compensation.

[0221] A composition containing the peptide ligand of the present invention or a cocktail thereof may be administered for prophylactic and / or therapeutic treatment. For some therapeutic uses, an amount suitable for achieving at least partial inhibition, suppression, modulation, apoptosis, or some other measurable parameter of a selected cell population is defined as the “therapeutic effective dose.” The amount required to achieve the above dose will vary depending on the severity of the disease and the general condition of the patient’s own immune system, but a general range is 0.005 to 5.0 mg of the selected peptide ligand per kilogram of body weight, and a dose of 0.05 to 2.0 mg / kg / dose is more commonly used. For prophylactic use, a composition containing the peptide ligand of the present invention or a cocktail thereof may also be administered at a similar or slightly lower dose.

[0222] A composition containing a peptide ligand according to the present invention may be used in preventive and therapeutic situations to assist in the alteration, inactivation, death, or elimination of a selected target cell population in mammals. Additionally, the peptide ligand described herein may be selectively used in vitro or in vitro to kill, deplete, or otherwise effectively eliminate a target cell population from a heterogeneous collection of cells. Blood from a mammal may be combined with the selected peptide ligand in vitro, thereby killing unintended cells or otherwise removing them from the mammal's blood according to standard techniques.

[0223] Therapeutic uses

[0224] According to a further aspect of the present invention, a heterotandem acyclic peptide complex as defined herein is provided for use in preventing, inhibiting, or treating cancer.

[0225] Examples of cancers (and their benign counterparts) that can be treated (or suppressed) are, without limitation, tumors of epithelial origin (various types of adenomas and carcinomas, e.g., adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, and other carcinomas), e.g., bladder and urinary tract, breast, gastrointestinal tract (including esophagus, stomach, small intestine, colon, rectum, and anus), liver (hepatocellular carcinoma), gallbladder and biliary system, exocrine pancreas, kidney, lung (e.g., adenocarcinoma, small cell lung carcinoma, non-small cell lung carcinoma, bronchoalveolar carcinoma, and mesothelioma), head and neck (e.g., tongue, oral cavity, larynx, pharynx, nasopharynx, tonsils, salivary glands, nasal cavity, and sinuses), ovaries, fallopian tubes, peritoneum, vagina, vulva, testes, cervix, uterine muscle, endometrium, thyroid (e.g., thyroid follicular carcinoma), adrenal glands, prostate, skin and adnexa (e.g., melanoma, basal cell carcinoma, squamous cell carcinoma, Carcinoma of keratoacanthoma, dysplastic nevus;Blood cancers (i.e., leukemia, lymphoma) and diseases of precancerous blood disorders and borderline cancers, including blood cancers of the lymphatic system and related conditions (e.g., acute lymphoblastic leukemia [ALL], chronic lymphoblastic leukemia [CLL], B-cell lymphomas, e.g., diffuse large B-cell lymphoma [DLBCL], follicular lymphoma, Burkitt lymphoma, mantle cell lymphoma, T-cell lymphoma and leukemia, natural killer [NK] cell lymphoma, Hodgkin lymphoma, hairy cell leukemia, monoclonal gammopathy of uncertain significance, plasmacytoma, multiple myeloma, and post-transplant lymphoproliferative disorders), and blood cancers of the myeloid system and related conditions (e.g., acute myeloid leukemia [AML], chronic myeloid leukemia [CML], chronic myelomonocytic leukemia [CMML], Hypereosinophilic syndrome, myeloproliferative disorders, e.g., polycythaemia vera, essential thrombocythaemia and primary myelofibrosis, myeloproliferative syndrome, myelodysplastic syndrome and promyelocyticleukemia; tumors of mesenchymal origin, e.g., sarcomas of soft tissue, bone or cartilage, e.g., osteosarcoma, fibrosarcoma, chondrosarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, Kaposi's sarcoma, Ewing sarcoma, synovial sarcoma, epithelioid sarcoma, gastrointestinal stromal tumors, benign and malignant histiocytomas, and dermatofibrosarcoma protuberans;Tumors of the central or peripheral nervous system (e.g., astrocytomas, gliomas and glioblastomas, meningiomas, ependymomas, pineal tumors and Schwannomas); endocrine tumors (e.g., pituitary tumors, adrenal tumors, islet cell tumors, parathyroid tumors, carcinoid tumors and medullary carcinoma of the thyroid); Tumors of the eye and adnexa (e.g., retinoblastoma); germ cell and trophoblastic tumors (e.g., teratomas, seminomas, dysgerminomas, hydatidiform moles, and choriocarcinomas); and pediatric and embryonic tumors (e.g., medulloblastoma, neuroblastoma, Wilms tumor, and primitive neuroectodermal tumors); or congenital or other syndromes that predispose the patient to cancer (e.g., Xeroderma Pigmentosum).;

[0226] In an additional embodiment, the cancer is selected from among blood cancers such as non-Hodgkin lymphoma (NHL), Burkitt lymphoma (BL), multiple myeloma (MM), B chronic lymphocytic leukemia (B-CLL), B and T acute lymphocytic leukemia (ALL), T cell lymphoma (TCL), acute myeloid leukemia (AML), hairy cell leukemia (HCL), Hodgkin lymphoma (HL), and chronic myeloid leukemia (CML).

[0227] In this document, the term "prevention" refers to the administration of a protective composition prior to the onset of disease. "Inhibition" refers to the administration of a composition after an inducing event but prior to the clinical manifestation of disease. "Treatment" refers to the administration of a protective composition after the manifestation of disease symptoms.

[0228] Animal model systems are available that can be used to screen the efficacy of peptide ligands in protection against or treatment of diseases. The use of said animal model systems is facilitated by the present invention, and the present invention allows for the development of polypeptide ligands capable of cross-reacting with human and animal targets in order to enable the use of animal models.

[0229] The present invention is further described below with reference to the following examples.

[0230] Examples

[0231] Example 1: Synthesis of Linker

[0232] COM128

[0233]

[0234] A mixture of Compound 1 (700.0 mg, 1.18 mmol, 1.0 eq), 3-azidopropane-1-amine (117.66 mg, 1.18 mmol, 1.0 eq), EDCI (270.4 mg, 1.41 mmol, 1.2 eq), and HOBt (190.6 mg, 1.41 mmol, 1.2 eq) was dissolved in DCM (20 mL, pre-degassed and purged with N2 three times), and the mixture was then stirred at 20–25°C for 1 hour under an N2 atmosphere. LC-MS confirmed that Compound 1 was completely eliminated and the target m / z (calculated MW: 677.33, observed m / z: 678.2 [M+H]) was achieved. + It was shown that a single main peak having )) was detected. The solvent was evaporated to produce compound 2 (600 mg, crude material) and obtained as a white solid.

[0235] A mixture of Compound 2 (600.0 mg, 885.3 μmol, 1.0 eq) and N-ethyl ethanolamine (1.29 g, 15.19 mmol, 1.50 mL, 17.2 eq) was dissolved in DCM (3 mL, pre-degassed and purged three times with N2), and the mixture was then stirred at 25–30°C for 2 hours under an N2 atmosphere. LC-MS confirmed that Compound 2 was completely extinguished and the target m / z (calculated MW: 455.51, observed m / z: 456.3 [M+H]) was achieved. + It was shown that a single main peak having )) was detected. The solvent was evaporated to produce compound 3 (400 mg, crude material) and obtained as a colorless oil.

[0236] A mixture of Compound 3 (150.0 mg, 329.3 μmol, 1.0 eq), Compound 4 (320.1 mg, 329.3 μmol, 1.0 eq), HATU (125.2 mg, 329.3 μmol, 1.0 eq), and DIEA (42.6 mg, 329.3 μmol, 57.4 μl, 1.0 eq) was dissolved in DMF (2 mL, pre-degassed and purged with N2 three times), and the mixture was then stirred at 25–30°C for 2 hours under an N2 atmosphere. LC-MS confirmed that Compound 3 was completely eliminated and the target m / z (calculated MW: 1408.76, observed m / z: 705.3 [M / 2+H]) was achieved. + It was shown that a single main peak having )) was detected. The solvent was evaporated to produce compound 5 (400 mg, crude material) and obtained as a yellow oil.

[0237] Compound 5 (400 mg, 283.77 μmol, 1.0 eq) was dissolved in DMF (4 mL, pre-degassed and purged three times with N2), then piperidine (862.2 mg, 10.13 mmol, 1 mL, 35.7 eq) was added, and the mixture was subsequently stirred at 25–30°C for 15 minutes under an N2 atmosphere. LC-MS confirmed that Compound 5 was completely eliminated and the target m / z (calculated MW: 1187.37, observed m / z: 594.4) [M / 2+H + ], 1187.4[M+H] + It was shown that a single main peak having )) was detected. The solvent was evaporated to produce COM128 (250 mg, crude substance) and obtained as a colorless oil.

[0238] COM129

[0239]

[0240] A mixture of Compound 1 (1.4 g, 1.47 mmol, 1.0 eq), 3-azidopropane-1-amine (162.1 mg, 1.62 mmol, 1.1 eq), EDCI (338.6 mg, 1.77 mmol, 1.2 eq), and HOBt (238.7 mg, 1.77 mmol, 1.2 eq) was dissolved in DCM (5 mL, pre-degassed and purged with N2 three times), and the mixture was then stirred at 20–25°C for 1 hour under an N2 atmosphere. LC-MS confirmed that Compound 1 was completely extinguished and the target m / z (calculated MW: 1033.14, observed m / z: 1033.2 [M+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was treated with a few drops of 1M HCl, and the solvent was removed by evaporating the organic layer under reduced pressure. Compound 2 (1.1 g, crude material) was obtained as a yellow oil.

[0241] A mixture of Compound 2 (1.1 g, 1.06 mmol, 1 eq) and N-ethyl ethanolamine (3.89 g, 53.24 mmol, 5.48 mL, 50 eq) was dissolved in DCM (5 mL, pre-degassed and purged three times with N2), and the mixture was then stirred at 20–25°C for 1 hour under an N2 atmosphere. LC-MS confirmed that Compound 2 was completely extinguished and the target m / z (calculated MW: 810.90, observed m / z: 810.9 [M+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was evaporated under reduced pressure and compound 3 (810 mg, crude material) was obtained as a white solid.

[0242] A mixture of Compound 3 (810.0 mg, 998.9 μmol, 1.0 eq), Compound 4 (810.7 mg, 1.10 mmol, 1.1 eq), HATU (455.8 mg, 1.20 mmol, 1.2 eq), and DIEA (258.2 mg, 2.00 mmol, 348.0 μl, 2.0 eq) was dissolved in DMF (2 mL, pre-degassed and purged with N2 three times), and the mixture was then stirred at 25–30°C for 2 hours under an N2 atmosphere. LC-MS confirmed that Compound 3 was completely eliminated and the target m / z (calculated MW: 1530.72, observed m / z: 765.5 [M / 2+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was treated with a few drops of 1M HCl, the organic layer was collected, and the solvent was removed by evaporation under reduced pressure. Compound 5 (1.1 g, crude material) was obtained as a yellow oil.

[0243] Compound 5 (1 g, 653.29 μmol, 1 eq) was dissolved in DCM (10 mL, pre-degassed and purged three times with N2), followed by the addition of piperidine (2.39 g, 32.66 mmol, 3.36 mL, 50 eq), and the mixture was then stirred at 25–30°C for 2 hours under an N2 atmosphere. LC-MS confirmed that Compound 5 was completely eliminated and the target m / z (calculated MW: 1308.47, observed m / z: 1308.4 [M+H]) was reached. + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (TFA conditions: Phase A: 0.075% TFA in H2O, Phase B: MeCN, Columns: Luna 200*25 mm 10 µm, C18, 110A and Gemin 150*30 mm, C18, 5 µm, 110A, connected, 50°C). COM129 (700 mg, 463.72 μmol, 70.98% yield) was obtained as a yellow solid.

[0244] COM130

[0245]

[0246] A mixture of Compound 1 (291 mg, 222.75 μmol, 1.0 eq), 3-azidopropane-1-amine (24.53 mg, 245.02 μmol, 1.1 eq), EDCI (51.24 mg, 267.30 μmol, 1.2 eq), and HOBt (36.12 mg, 267.30 μmol, 1.2 eq) was dissolved in DCM (3 mL, pre-degassed and purged with N2 three times), and the mixture was then stirred at 20–25°C for 1 hour under an N2 atmosphere. LC-MS confirmed that Compound 1 was completely extinguished and the target m / z (calculated MW: 1388.53, observed m / z: 694.7 [M / 2+H]) was achieved. + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (neutral conditions). Compound 2 (200 mg, 144.04 μmol, 64.66% yield) was obtained as a white solid.

[0247] A mixture of Compound 2 (200 mg, 144.04 μmol, 1.0 eq) and N-ethyl ethanolamine (210.7 mg, 2.88 mmol, 297 μl, 20.0 eq) was dissolved in DCM (3 mL, pre-degassed and purged three times with N2), and the mixture was then stirred at 20–25°C for 1 hour under an N2 atmosphere. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (calculated MW: 1166.29, observed m / z: 1166.3 [M+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was evaporated and compound 3 (150 mg, crude material) was obtained as a yellow oil.

[0248] A mixture of Compound 3 (150 mg, 128.61 μmol, 1.0 eq), Compound 4 (75 mg, 144.91 μmol, 1.13 eq), HATU (58.7 mg, 154.34 μmol, 1.2 eq), and DIEA (33.24 mg, 257.23 μmol, 44.80 μl, 2.0 eq) was dissolved in DMF (5 mL, pre-degassed and purged with N2 three times), and the mixture was then stirred at 20–25°C for 2 hours under an N2 atmosphere. LC-MS confirmed that Compound 3 was completely eliminated and the target m / z (calculated MW: 1665.84, observed m / z: 833.2 [M / 2+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was removed under reduced pressure and compound 5 (300 mg, crude material) was obtained as a yellow oil.

[0249] Crude compound 5 (300 mg, dissolved in 10 mL DMF) was added to piperidine (2 mL), and the mixture was stirred at 30°C for 2 hours. LCMS was set to the target m / z (MW: 1443.60, observed m / z: 722.7 [M / 2+H]). +It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (neutral conditions). COM130 (140 mg, 58.19 μmol, 32.31% yield, 60% purity) was obtained as a white solid.

[0250] COM131

[0251]

[0252] A mixture of Compound 1 (700.0 mg, 1.18 mmol, 1.0 eq), 3-azidopropane-1-amine (117.7 mg, 1.18 mmol, 1.0 eq), HOBt (190.6 mg, 1.41 mmol, 1.2 eq), and EDCI (270.4 mg, 1.41 mmol, 1.2 eq) was dissolved in DCM (20 mL, pre-degassed and purged with N2 three times), and the mixture was then stirred at 25–30°C for 2 hours under an N2 atmosphere. LC-MS confirmed that Compound 1 was completely extinguished and the target m / z (calculated MW: 677.75, observed m / z: 678.2 [M+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was treated with a few drops of 1M HCl, the organic layer was collected, and evaporated under reduced pressure. Compound 2 (600.0 mg, crude material) was obtained as a white solid.

[0253] Compound 2 (600.0 mg, 885.2 μmol, 1.0 eq) was dissolved in DMF (3 mL, pre-degassed and purged three times with N2), followed by the addition of piperidine (1.29 g, 15.19 mmol, 1.50 mL, 17.2 eq), and the mixture was stirred at 25–30°C for 2 hours under an N2 atmosphere. LC-MS confirmed that Compound 2 was completely extinguished and the target m / z (calculated MW: 455.51, observed m / z: 456.3 [M+H]) was achieved. +It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions), and compound 3 (400.0 mg, 879.1 μmol) was obtained as a colorless oil.

[0254] A mixture of Compound 3 (250.0 mg, 548.83 μmol, 1.0 eq), Compound 4 (284.1 mg, 548.83 μmol, 1.0 eq), HATU (229.6 mg, 603.72 μmol, 1.1 eq), and DIEA (141.9 mg, 1.10 mmol, 191.19 μl, 2.0 eq) was dissolved in DCM (20 mL, pre-degassed and purged with N2 three times), and the mixture was then stirred at 25–30°C for 2 hours under an N2 atmosphere. LC-MS confirmed that Compound 3 was completely eliminated and the target m / z (calculated MW: 955.06, observed m / z: 955.6 [M+H]) was achieved. + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (under TFA conditions). Compound 5 (400.0 mg, 419.1 μmol) was obtained as a white solid. Compound 5 (400.0 mg, 418.82 μmol, 1.0 eq) was dissolved in DMF (4 mL, pre-degassed and purged three times with N2), followed by the addition of piperidine (862.2 mg, 10.13 mmol, 1 mL, 24.2 eq), and the mixture was stirred at 25–30°C for 2 hours under an N2 atmosphere. LC-MS showed that Compound 5 was completely eliminated and the target m / z (calculated MW: 732.83, observed m / z: 733.3 [M+H] + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (TFA conditions). COM131 (200 mg, 272.9 μmol) was obtained as a colorless oil.

[0255] COM470

[0256]

[0257] HATU (85.40 mg, 224.75 μmol, 1.5 eq) and DIEA (19.37 mg, 149.83 μmol, 26.10 μl, 1.0 eq) were added to a solution of COM122 (228 mg, 149.83 μmol, 1.0 eq) and Compound 1 (51.31 mg, 164.82 μmol, 1.1 eq) in DMF (6 mL). The mixture was stirred at 25–30°C for 2 hours. LC-MS confirmed that Compound 1 was completely quenched and the target m / z (MW: 1814.99, observed m / z: 908.2 [M / 2+H]) was reached. + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (neutral conditions). Compound 2 (54 mg, 29.75 μmol, 19.86% yield) was obtained as a white solid.

[0258] Piperidine (61 mg, 715 μmol, 71 μl, 24.0 eq) was added to a solution of Compound 2 (54 mg, 29.8 μmol, 1.0 eq) in DMF (2 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 1592.75, observed m / z: 796.27 [M / 2+H]) was reached. + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). COM470 (40 mg, 25.11 μmol, 84.41% yield) was obtained as a white solid.

[0259] COM471

[0260]

[0261] A mixture of Compound 1 (900 mg, 1.23 mmol, 1.0 eq) and Compound 2 (1.0 g, 3.21 mmol, 2.6 eq) was dissolved in DCM (20 mL), and then (284.0 mg, 1.48 mmol, 1.2 eq) and HOBt (200.2 mg, 1.48 mmol, 1.2 eq) were added. The mixture was stirred at 25°C for 2 hours. LC-MS confirmed that Compound 1 was completely extinguished and the target m / z (calculated MW: 1021.49, observed m / z: 1022.2 [M+H]) was reached. + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by pre-HPLC (TFA conditions). Compound 3 (0.900 g, 880.53 μmol, 71.30% yield) was obtained as a white solid.

[0262] A mixture of Compound 3 (500.0 mg, 489.19 μmol, 1.0 eq) and Compound 4 (257.6 mg, 489.19 μmol, 1.0 eq) was dissolved in DCM (5 mL), and then HOBt (132.2 mg, 978.37 μmol, 2.0 eq) and EDCI (187.6 mg, 978.37 μmol, 2.0 eq) were added. The mixture was stirred at 25–30°C for 2 hours. LC-MS confirmed that Compound 3 was completely extinguished and the target m / z (MW: 1529.80, observed m / z: 765.9 [M / 2+H]) was reached. + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (neutral conditions). Compound 3 (420 mg, 246.94 μmol, 50.48% yield) was produced and obtained as a colorless oil.

[0263] Compound 5 (420 mg, 274.38 μmol, 1.0 eq) was dissolved in DMF (4 mL), and then piperidine (865.2 mg, 10.16 mmol, 1 mL, 37 eq) was added. The mixture was stirred at 25–30°C for 2 hours. LC-MS confirmed that Compound 5 was completely extinguished and the target m / z (calculated MW: 1308.48, observed m / z: 654.8 [M / 2+H]) was reached. + It was shown that a single main peak having )) was detected. The crude product was purified by pre-HPLC (TFA conditions). COM471 (386 mg, 265.50 μmol, 96.76% yield) was obtained as a colorless oil.

[0264] COM472

[0265]

[0266] A mixture of Compound 1 (0.5 g, 839.43 μmol, 1.0 eq), Compound 2 (627.0 mg, 839.43 μmol, 1.0 eq), and DIEA (217.0 mg, 1.68 mmol, 292.4 μL, 2.0 eq) was dissolved in DMF (2 mL), and then HATU (319.2 mg, 839.4 μmol, 1.0 eq) was added to the mixture. The mixture was then stirred at 25°C for 30 minutes. TLC (DCM:CH3OH=10:1, R f =0.24) showed that compound 1 was completely extinguished and a new spot was formed. By evaporating the solvent, compound 3 (0.45 g, 339.75 μmol, 40.47% yield, crude material) was obtained as a colorless oil and used in the next step without further purification.

[0267] Compound 3 (450.0 mg, 339.75 μmol, 1.0 eq) was dissolved in DMF (8 mL), and then piperidine (2 mL) was added. The mixture was stirred at 25°C for 15 minutes. LC-MS showed that Compound 3 was completely eliminated and the target (calculated MW: 1102.27, observed m / z: 552.1 ([M / 2+H]) + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (TFA conditions). Compound 3 (370.0 mg, 335.67 μmol, 98.80% yield) was obtained as a colorless oil.

[0268] HATU (31 mg, 81.68 μmol, 1.5 eq) and DIEA (10.5 mg, 61.68 μmol, 15 μl, 1.5 eq) were added to a solution of COM126 (60 mg, 54.45 μmol, 1.0 eq) and Compound 4 (15.5 mg, 81.68 μmol, 1.5 eq) in DMF (5 mL). The mixture was stirred at 30°C for 2 hours. LC-MS showed that COM126 was completely eliminated and a single target main peak was detected. The mixture was evaporated to remove the solvent, and Compound 5 (30 mg, crude material) was obtained as a colorless oil, which was used in the next step without further purification.

[0269] Compound 5 (30 mg, 23.57 μmol, 1.0 eq) was dissolved in DCM (4.5 mL), followed by the addition of TFA (0.5 mL), and the mixture was stirred at 25-30°C for 2 hours. LC-MS showed that Compound 5 was completely eliminated and a single target main peak was detected. The residue was purified by pre-HPLC (TFA conditions). COM472 (10 mg, 8.52 μmol) was obtained as a white solid.

[0270] COM473

[0271]

[0272] A mixture of Compound 1 (300 mg, 449.96 μmol, 1.0 eq), Compound 2 (138 mg, 449.96 μmol, 1.0 eq), HOBt (122 mg, 899.93 μmol, 2.0 eq), and EDCI (173 mg, 899.93 μmol, 2.0 eq) was dissolved in DCM (10 mL, pre-degassed and purged three times with N2), and the mixture was then stirred at 20–25°C for 1 hour under an N2 atmosphere. LC-MS confirmed that Compound 1 was completely eliminated and the target (MW: 955.06, observed m / z: 955.3 [M+H]) + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent. The mixture was evaporated under reduced pressure and compound 3 (300 mg, crude material) was obtained as a yellow oil.

[0273] Compound 3 (300 mg, 314.12 μmol, 1.0 eq) was dissolved in DMF (4 mL), followed by the addition of piperidine (1 mL), and the mixture was stirred at 20-25°C for 1 hour. LC-MS confirmed that Compound 3 was completely eliminated and the target m / z (MW: 732.83, observed m / z: 733.2 [M+H]) was achieved. + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (neutral conditions). COM473 (160 mg, 218.33 μmol, 69.51% yield) was obtained as a colorless oil.

[0274] Example 2: Synthesis of EphA2 / CD137-linked heterotandem acyclic peptide

[0275] BCY9173

[0276]

[0277] Manufacturing process of BCY9172-PEG12-N3

[0278]

[0279] BCY9172 (520 mg, 248.16 μmol, 1 eq) and Compound 1 (370 mg, 499.47 μmol, 2.01 eq) were dissolved in DMF (5 mL), DIEA (48.11 mg, 372.24 μmol, 64.84 μl, 1.5 eq) was added, and the mixture was then stirred at 30°C for 12 hours. LC-MS confirmed that BCY9172 was completely eliminated and the target m / z (calculated MW: 2721.12, observed m / z: 1360.9 [M / 2+H]) was reached. + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (284 mg, 101.10 μmol, 40.74% yield, 96.87% purity) was obtained as a white solid.

[0280]

[0281] BCY9173 manufacturing process

[0282] The reaction was carried out in parallel in two independent vessels. For one vessel, Compound 2 (100 mg, 36.75 μmol, 1.0 eq) and BCY6169 (120 mg, 36.78 μmol, 1.0 eq) were first dissolved in 10 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 91.9 μL, 1.0 eq), VcNa (0.4 M, 183.8 μL, 2.0 eq), and THPTA (0.4 M, 91.9 μL, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 40°C for 16 hours under an N2 atmosphere. LC-MS showed that Compound 2 was completely extinguished and the target m / z (calculated MW: 5983.85, observed m / z: 997.6600 ([M / 6+H]) + ) and 1197.2300([M / 5+H] +It was shown that a single main peak having ) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY9173 (218 mg, 34.97 μmol, 47.58% yield, 96% purity) was obtained as a white solid.

[0283] BCY7985

[0284]

[0285] General manufacturing process of BCY7859

[0286]

[0287] EDCI (89.3 mg, 466 μmol) was added to a solution of N3-PEG12-COOH (250 mg, 388 μmol) and HOSu (67.0 mg, 583 μmol) in DMA (4.5 mL) and DCM (1.5 mL) while stirring at 20°C for 16 hours. DIEA (186 mg, 1.44 mmol, 250 μl) was added to another 50 mL round-bottom flask containing a mixture of BCY7732 (855 mg, 388 μmol) in 5 mL of DMA while stirring for 10 minutes. Subsequently, the initial reaction mixture was added to the flask while stirring at 20°C for an additional 5 hours. LC-MS (ES8396-307-P1B1) showed that BCY7732 was completely eliminated and a single main peak with the target mass was detected. The resulting reaction mixture was directly purified by pre-HPLC (TFA conditions) to provide compound BCY7859 (621 mg, 200 μmol, 51.6% yield, TFA salt) as a white solid.

[0288] General manufacturing process of BCY6169

[0289]

[0290] DIEA (36.6 mg, 283 μmol, 49.3 μl) was added to a solution of BCY6099 (300 mg, 94.3 μmol) in DMA (2 mL) while stirring for 10 minutes. Afterwards, PYA-NHS (36.8 mg, 189 μmol) was added while stirring for an additional 15 hours at 20°C. LC-MS showed that BCY6099 was completely eliminated and a single main peak with the desired mass was detected. The reaction mixture was purified by pre-HPLC (neutral conditions) to yield compound BCY6169 (299 mg, 86.2 μmol, 91.5% yield) as a white solid.

[0291] General manufacturing process of BCY7985

[0292]

[0293] Aqueous ascorbic acid solution (0.8 M, 963 µL) was added to a solution of BCY7859 (220 mg, 77.8 μmol) and BCY6169 (251 mg, 77.1 μmol) in DMF (5 mL) purged with nitrogen for 2 hours, followed by the addition of aqueous CuSO4 (0.8 M, 289 µL) under a nitrogen atmosphere. The mixture was then stirred at 20°C for 2 hours. LC-MS showed that BCY6169 was completely eliminated and a single main peak with the desired mass was detected. The reaction mixture was directly purified by pre-HPLC (under TFA conditions) to yield compound BCY7985 (283 mg, 43.4 μmol, 56.3% yield, TFA) as a white solid.

[0294] BCY8942

[0295]

[0296] General manufacturing process of BCY8940

[0297]

[0298] HOSu (32.2 mg, 280 μmol, 1.5 eq) was added while stirring to a solution of N3-PEG12-COOH (120 mg, 186 μmol, 1.0 eq) in DMA (3 mL) and DCM (1 mL). Then, EDCI (42.9 mg, 224 μmol, 1.2 eq) was added to the mixture while stirring for an additional 7 hours at 20°C. LCMS showed that the activated ester was fully formed. DIEA (120 mg, 932 μmol, 162 μl, 5.0 eq) was added while stirring to another flask containing BCY8045 (410 mg, 186 μmol, 1.0 eq) in DMA (3 mL), followed by the addition of the activated ester, and the mixture was stirred at 20°C for 18 hours. LC-MS showed that a single main peak with the desired m / z was detected. The reaction mixture was concentrated under vacuum to remove DCM. The resulting mixture was purified by pre-HPLC (TFA conditions) to provide BCY8940 (190 mg, 67.2 μmol, 36.1% yield) as a white solid.

[0299] General manufacturing process of BCY8942

[0300]

[0301] (2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (1.0 M, 92.0 µL) and CuSO4 (1.0 M, 27.6 µL) were added to a solution of BCY8940 (28.6 mg, 10.1 μmol, 1.1 eq) and BCY6169 (30.0 mg, 9.19 μmol, 1.0 eq) in DMF (2.0 mL) while stirring under a nitrogen atmosphere at 20°C for 2 hours. LC-MS confirmed that BCY6169 was completely eliminated and the target m / z (calculated MW: 6089.91 observed) m / z : 1218.4([M / 5+H] + ), 1016.0([M / 6+H] + ), 870.7([M / 7+H]+ It was shown that a single main peak having ) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) to provide compound BCY8942 (15.4 mg, 2.46 μmol, 26.8% yield, 97.3% purity) as a white solid.

[0302] BCY8943

[0303]

[0304] General manufacturing process of BCY8941

[0305]

[0306] DIEA (12.8 mg, 98.7 μmol, 17.2 μl) was added to a solution of BCY6015 (a peptide identical to BCY8941 except for the absence of the PYA moiety; 100 mg, 32.9 μmol) in DMA (2 mL) while stirring for 10 minutes. Subsequently, (2,5-dioxopyrrolidine-1-yl)pent-4-inoate (12.8 mg, 65.8 μmol) was added to the mixture, followed by further stirring at 20°C for 16 hours. LC-MS confirmed that Compound 1 was completely extinguished and the target m / z (calculated MW: 3119.60, observed m / z : 1040.5([M / 3+H] + It was shown that a single main peak having ) was detected. The mixture was purified by pre-HPLC (neutral conditions) to provide compound BCY8941 (90.0 mg, 28.9 μmol, 87.7% yield) as a white solid.

[0307] General manufacturing process of BCY8943

[0308]

[0309] (2R)-2[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (1.0 M, 270 µL) and CuSO4 (1.0 M, 80.9 µL) were added to a solution of BCY7859 (can be prepared as described in BCY7985; 40.0 mg, 14.2 μmol) and BCY8941 (42.0 mg, 13.5 μmol) in DMSO (2 mL, pre-purged with nitrogen for 1 hour). The mixture was purged with nitrogen three times and stirred at 15°C for 2 hours. LC-MS confirmed that BCY8941 was completely eliminated and the target m / z (calculated MW: 5946.77, observed m / z : 1190.2([M / 5+H] + ), 991.5([M / 6+H] + ), 849.9([M / 7+H] + It was shown that a single main peak having ) was detected. The reaction mixture was purified by pre-HPLC (A: 0.075% TFA in H2O, B: ACN) to provide compound BCY8943 (11.5 mg, 1.90 μmol, 14.1% yield, 98.1% purity) as a white solid.

[0310] BCY9647

[0311]

[0312] Preparation process of Compound 2

[0313]

[0314] TEA (8.65 mg, 11.9 μL, 1.5 eq) was added to a solution of COM134 (30.0 mg, 57.0 μmol, 1.0 eq) and Compound 1 (17.2 mg, 85.3 μmol, 1.5 eq) in DCM (0.5 mL). The mixture was stirred at 25°C for 1 hour. LC-MS confirmed that COM134 was completely eliminated and the target mass (calculated MW: 691.72, observed m / z : 692.3([M+H] + ) and 709.3([M+NH4]+ It was shown that a single main peak having ) was detected. The reaction mixture was concentrated under reduced pressure and then freeze-dried to produce crude compound 2 (30.5 mg, crude substance) as white.

[0315] Preparation process of Compound 3

[0316]

[0317] BCY6099 (46 mg, 1.0 eq) and DIEA (5.61 mg, 7.55 µl, 3.0 eq) were added to a solution of Compound 2 (10 mg, 1.0 eq) in DMF (1 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (calculated MW: 3735.28 observed) was reached. m / z : 1245.9([M / 3+H] + ) and 934.5([M / 4+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (34 mg, 62.96% yield, 100% purity) was obtained as a white solid.

[0318] BCY9647 manufacturing process

[0319]

[0320] A mixture of compound 3 (34 mg, 9.10 μmol, 1.0 eq), BCY7741 (23 mg, 10.08 μmol, 1.11 eq), and THPTA (0.4 M, 11.4 μl, 0.5 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 11.4 μl, 0.5 eq) and VcNa (0.4 M, 22.8 μl, 1 eq) were added under N2. The pH of the solution was adjusted to 8 by titrating 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25–30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z (calculated MW: 6016.82, observed m / z : 1204.1([M / 5+H] + ), 1003.5([M / 6+H] + ), 860.3([M / 7+H] + It was shown that a single main peak having ) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9647 (31.2 mg, 54.67% yield, 95.96% purity) was obtained as a white solid.

[0321] BCY9648

[0322]

[0323] Preparation process of Compound 2

[0324]

[0325] TEA (4.14 mg, 40.94 μmol, 5.70 μl, 1.5 eq) was added to a solution of COM135 (30 mg, 27.29 μmol, 1.0 eq) and Compound 1 (8.25 mg, 40.94 μmol, 1.5 eq) in DCM (0.5 mL). The mixture was stirred at 25-30°C for 1 hour. LC-MS confirmed that COM135 was completely eliminated and the target mass [Calculated MW: 1264.41, Observed m / z : 1281.4([M+NH4] + ), 649.8([M / 2+H] + It was shown that a single main peak having )] was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions) to provide compound 2 (18 mg, 14.2 μmol, 52.14% yield).

[0326] Preparation process of Compound 3

[0327]

[0328] BCY6099 (23 mg, 7.23 μmol, 1.02 eq) and DIEA (2.76 mg, 21.35 μmol, 3.72 μl, 3.0 eq) were added to a solution of Compound 3 (9 mg, 7.12 μmol, 1 eq) in DMF (1 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely extinguished and the target m / z (calculated MW: 4307.96 observed) was achieved. m / z : 1436.9([M / 3+H] + ), 1077.9([M / 4+H] + ), 862.5([M / 5+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (14.6 mg, 47.61% yield, 100% purity) was obtained as a white solid.

[0329] BCY9648 manufacturing process

[0330]

[0331] A mixture of Compound 3 (14.6 mg, 3.39 μmol, 1 eq), BCY7741 (8.5 mg, 3.73 μmol, 1.1 eq), and THPTA (0.4 M, 4.3 μl, 0.5 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 4.3 μl, 0.5 eq) and VcNa (0.4 M, 8.6 μl, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred under an N2 atmosphere at 25-30°C for 12 hours. LC-MS showed that Compound 3 was completely extinguished and the target m / z (calculated MW: 6589.50, observed m / z : 1098.8([M / 6+H] + ), 942.1([M / 7+H] + ), 824.6([M / 8+H] + It was shown that a single main peak having ) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9648 (14.7 mg, 63.34% yield, 96.22% purity) was obtained as a white solid.

[0332] BCY9655

[0333]

[0334] Preparation process of Compound 2

[0335]

[0336] TEA (15.34 mg, 151.59 μmol, 21.10 μl, 1.5 eq) was added to a solution of COM128 (120 mg, 101.06 μmol, 1.0 eq) and Compound 1 (25 mg, 124.03 μmol, 1.25 eq) in DCM (0.5 mL). The mixture was stirred at 25°C for 1 hour. LC-MS was performed at the target m / z (calculated MW: 1352.48, observed m / z : 676.8([M / 2+H] + ), 1369.3([M+NH4] + It was shown that a single main peak having ) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (neutral conditions). Compound 2 (14 mg, 8.99 μmol, 8.90% yield, 86.86% purity) was obtained as a colorless oil.

[0337] Preparation process of Compound 3

[0338]

[0339] DIEA (2.01 mg, 15.53 μmol, 2.70 μL) was added to a solution of Compound 2 (7 mg, 5.18 μmol, 1.0 eq) and BCY6099 (16 mg, 5.03 μmol, 1.0 eq) in DMF (2 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (calculated MW: 4396.02, observed m / z : 879.8([M / 5+H] + ) and 1099.8([M / 4+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (under 0.1% TFA conditions). Compound 3 (11.8 mg, 48.29% yield, 93.11% purity) was obtained as a white solid.

[0340] BCY9655 manufacturing process

[0341]

[0342] A mixture of compound 3 (11.8 mg, 2.69 μmol, 1.0 eq), BCY7741 (7.0 mg, 3.07 μmol, 1.14 eq), and THPTA (0.4 M, 6.8 μl, 1 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 6.8 μl, 1.0 eq) and VcNa (0.4 M, 13.6 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25–30°C for 12 hours under an N2 atmosphere. LC-MS confirmed that Compound 3 was completely extinguished and the target m / z (calculated MW: 6677.57, observed m / z : 1113.7([M / 6+H] + ), 954.7([M / 7+H] + It was shown that a single main peak having )) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9655 (1.9 mg, 0.26 μmol, 9.65% yield, 91.15% purity) was obtained as a white solid.

[0343] BCY9656

[0344]

[0345] Preparation process of Compound 2

[0346]

[0347] TEA (3.5 mg, 34.39 μl, 4.8 μl, 1.5 eq) was added to a solution of COM129 (30.0 mg, 22.93 μmol, 1.0 eq) and Compound 1 (6.9 mg, 34.39 μmol, 1.5 eq) in DCM (3 mL). The mixture was degassed and purged three times with N2, followed by stirring the mixture under an N2 atmosphere at 25°C for 1 hour. LC-MS confirmed that COM129 was completely eliminated and the target m / z (calculated MW: 1473.58, observed m / z : 737.3([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (neutral conditions) to produce compound 2 (12.3 mg, 8.35 μmol, 36.41% yield) as a white solid.

[0348] Preparation process of Compound 3

[0349]

[0350] TEA (0.7 mg, 6.93 μmol, 1 μL, 1.1 eq) was added to a solution of Compound 2 (9.26 mg, 6.28 μmol, 1.0 eq) and BCY6099 (10 mg, 3.14 μmol, 0.5 eq) in DMF (3 mL). The mixture was degassed and purged with N2 three times, followed by stirring the mixture under an N2 atmosphere at 25-30°C for 1 hour. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (calculated MW: 4517.12, observed m / z : 1129.8([M / 4+H] + ), 904.1([M / 5+H] + ), 753.7([M / 6+H] +It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (12 mg, 72.36% yield, 85.58% purity) was obtained as a white solid.

[0351] BCY9656 manufacturing process

[0352]

[0353] A mixture of compound 3 (11 mg, 2.44 μmol, 1.0 eq), BCY7741 (6.0 mg, 2.63 μmol, 1.08 eq), and THPTA (0.4 M, 6.1 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 6.1 μl, 1.0 eq) and VcNa (0.4 M, 12.2 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by titrating 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25–30°C for 12 hours under an N2 atmosphere. LC-MS indicated that Compound 3 was completely extinguished and the target m / z (calculated MW: 6798.66, observed m / z : 1133.8([M / 6+H] + ), 971.9([M / 7+H] + ), 850.7([M / 8+H] + It was shown that a single main peak having ) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9656 (6.8 mg, 37.36% yield, 90.97% purity) was obtained as a white solid.

[0354] BCY9657

[0355]

[0356] Preparation process of Compound 2

[0357]

[0358] TEA (3.2 mg, 31.17 μmol, 4.4 μL, 1.5 eq) was added to a solution of COM130 (30.0 mg, 20.78 μmol, 1.0 eq) and Compound 1 (6.3 mg, 31.17 μmol, 1.5 eq) in DCM (3 mL). The mixture was stirred at 25-30°C for 1 hour. LC-MS confirmed that COM130 was completely extinguished and the target m / z (calculated MW: 1608.7, observed m / z : 804.8([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure and freeze-dried to obtain compound 2 (7.9 mg, crude material) as a white solid.

[0359] Preparation process of Compound 3

[0360]

[0361] DIEA (1.9 mg, 14.73 μmol, 2.6 μL, 3.0 eq) was added to a solution of Compound 2 (7.9 mg, 4.91 μmol, 1.0 eq) and BCY6099 (16 mg, 5.03 μmol, 1.02 eq) in DMF (1 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely extinguished and the target m / z (calculated MW: 4652.25, observed m / z : 1551.3([M / 3+H] + ), 1163.6([M / 4] + ), 931.1([M / 5+H] + ), 776.1([M / 6+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (13.3 mg, 2.86 μmol, 53.22% yield, 91.42% purity) was obtained as a white solid.

[0362] BCY9657 manufacturing process

[0363]

[0364] A mixture of compound 3 (13.3 mg, 2.86 μmol, 1.0 eq), BCY7741 (7.0 mg, 3.07 μmol, 1.03 eq), and THPTA (0.4 M, 7.5 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 7.5 μl, 1 eq) and VcNa (0.4 M, 15 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by titrating 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 6933.78, observed m / z : 1156.7([M / 6+H] + ), 991.4([M / 7+H] + ), 867.4([M / 8+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9657 (8.4 mg, 40.21% yield, 94.9% purity) was obtained as a white solid.

[0365] BCY9658

[0366]

[0367] Preparation process of Compound 2

[0368]

[0369] TEA (36.4 mg, 359.23 μmol, 50.0 μl, 1.6 eq) was added to a solution of COM131 (167.0 mg, 227.89 μmol, 1.0 eq) and Compound 1 (55.0 mg, 272.87 μmol, 1.2 eq) in DCM (5 mL). The mixture was stirred at 25-30°C for 1 hour. LC-MS was performed at the target m / z (MW: 897.93) and the observed value was 920.3 ([M+Na + It was shown that a single main peak having ]) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (35 mg, 33.74 μmol, 14.81% yield, 86.56% purity) was obtained as a colorless oil.

[0370] Preparation process of Compound 3

[0371]

[0372] DIEA (6.48 mg, 65.05 μmol, 50.1 μl, 4.0 eq) was added to a solution of Compound 2 (15 mg, 16.71 μmol, 1.0 eq) and BCY6099 (53 mg, 16.65 μmol, 1.0 eq) in DMF (2 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 3941.47) was observed. m / z : 986.0([M / 4+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). [BCY6099]-[COM131] (5 mg, 50.48% yield, 94.96% purity) was obtained as a white solid.

[0373] BCY9658 manufacturing process

[0374]

[0375] A mixture of compound 3 (35 mg, 8.88 μmol, 1.0 eq), BCY7741 (21 mg, 9.20 μmol, 1.03 eq), and THPTA (0.4 M, 22.2 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 22.2 μl, 1.0 eq) and VcNa (0.4 M, 44.4 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 6223.01] was observed. m / z : 1038.0([M / 6+H] + ) and 889.8([M / 8+H] + It was shown that a single main peak having )) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9658 (13.2 mg, 21.54% yield, 90.16% purity) was obtained as a white solid.

[0376] BCY9659

[0377]

[0378] Preparation process of Compound 2

[0379]

[0380] TEA (36.4 mg, 359.23 μmol, 50 μL, 5.5 eq) was added to a solution of COM132 (20.0 mg, 65.28 μmol, 1.0 eq) and Compound 1 (15.8 mg, 78.34 μmol, 1.2 eq) in DCM (5 mL). The mixture was stirred at 25°C for 1 hour. LC-MS was performed at the target m / z (MW: 471.46, observed m / z : 489.2([M+NH4] +It was shown that a single main peak having ) was detected. The reaction mixture was concentrated under reduced pressure to provide compound 2 (26 mg, crude material) as a colorless oil.

[0381] Preparation process of Compound 3

[0382]

[0383] TEA (0.7 mg, 6.93 μmol, 1 μL, 1.5 eq) was added to a solution of Compound 2 (15.0 mg, 4.71 μmol, 1.0 eq) and BCY6099 (3.33 mg, 7.07 μmol, 1.5 eq) in DMF (3 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 3515.01, observed m / z : 1172.1([M / 3+H] + ) 879.5([M / 4+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (12.7 mg, 3.26 μmol, 69.23% yield, 90.3% purity) was obtained as a white solid.

[0384] BCY9659 manufacturing process

[0385]

[0386] A mixture of compound 3 (12.7 mg, 2.89 μmol, 1.0 eq), BCY7741 (6.80 mg, 2.98 μmol, 1.03 eq), and THPTA (1.3 mg, 2.99 μmol, 1.03 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 7.3 μL, 1.0 eq) and VcNa (0.4 M, 14.6 μL, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 5796.54] was observed. m / z : 1159.8([M / 5+H]) 966.7([M / 6+H] + It was shown that a single main peak having )) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9659 (6.2 mg, 1.06 μmol, 36.58% yield, 98.86% purity) was obtained as a white solid.

[0387] BCY9758

[0388]

[0389] Preparation process of Compound 2

[0390]

[0391] DIEA (0.9 mg, 7.07 μmol, 1.2 μL, 2.0 eq) was added to a solution of Compound 1 (5.0 mg, 3.54 μmol, 1.0 eq) and BCY6099 (11.3 mg, 3.54 μmol, 1.0 eq) in DMF (3 mL). The mixture was stirred at 25-30°C for 20 minutes. LC-MS was performed at the target m / z (MW: 4481.11, observed m / z : 1101.3([M / 4+H] +It was shown that a single main peak having )) was detected. The reaction mixture was filtered, concentrated under reduced pressure, and freeze-dried to provide compound 2 (15 mg, crude material) as a white solid.

[0392] BCY9758 manufacturing process

[0393]

[0394] DIEA (0.9 mg, 7.07 μmol, 1.2 μL, 2.1 eq) was added to a solution of Compound 2 (15 mg, 3.35 μmol, 1.0 eq) and BCY7732 (14.74 mg, 6.69 μmol, 2.0 eq) in DMF (3 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 6567.48, observed m / z : 1095.1([M / 6+H] + ), 938.8([M / 7+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). BCY9758 (5.8 mg, 24.26% yield, 91.97% purity) was obtained as a white solid.

[0395] BCY10568

[0396]

[0397] Manufacturing process of BCY8919-PEG12-N3

[0398]

[0399] BCY8919 (80.0 mg, 38.47 μmol, 1.0 eq) and Compound 1 (29.6 mg, 40.01 μmol, 1.04 eq) were dissolved in DMSO (1 mL). Subsequently, DIPEA (7.46 mg, 55.71 μmol, 10.0 μl, 1.5 eq) was added to the solution, and the mixture was stirred at 25–30°C for 2 hours. LC-MS confirmed that most of the BCY8919 had been eliminated and the target m / z (calculated MW: 2705.16, observed m / z: 1353.1 [M / 2+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (18.6 mg, 6.86 μmol, 17.83% yield, 99.76% purity) was obtained as a white solid.

[0400] BCY10568 manufacturing process

[0401]

[0402] Compound 2 (9.0 mg, 3.33 μmol, 1.0 eq) and BCY6169 (11.0 mg, 3.36 μmol, 1.01 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 8.3 μl, 1.0 eq), VcNa (1.4 mg, 7.06 μmol, 2.1 eq), and THPTA (1.4 mg, 3.22 μmol, 1.0 eq). Finally, 0.4 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 16 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 5967.90, observed m / z: 995.00 ([M / 5+H]) + ) and 1194.70([M / 6+H] +It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY10568 (13.4 mg, 2.16 μmol, 69.44% yield, 96.3% purity) was obtained as a white solid.

[0403] BCY10570

[0404]

[0405] Manufacturing process of BCY8920-PEG12-N3

[0406]

[0407] DIEA (3.36 mg, 25.96 μmol, 4.5 μl, 1.5 eq) was added to a solution of BCY8920 (37 mg, 17.31 μmol, 1.0 eq) and Compound 1 (15 mg, 20.25 μmol, 1.2 eq) in DMSO (2 mL). The mixture was stirred at 30°C for 12 hours. LC-MS confirmed that BCY8920 was completely eliminated and the target m / z (calculated MW: 2763.2, observed m / z : 689.07([M / 4-H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by pre-HPLC (neutral conditions). Compound 2 (22.8 mg, 8.15 μmol, 47.09% yield, 98.78% purity) was provided as a white solid.

[0408] BCY10570 manufacturing process

[0409]

[0410] Compound 2 (6 mg, 2.17 μmol, 1.0 eq) and BCY6169 (7.08 mg, 2.17 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 5.4 µl, 1.0 eq), VcNa (0.4 M, 10.8 µl, 2.0 eq), and THPTA (0.4 M, 5.4 µl, 1.0 eq). Finally, 0.2 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 4 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (MW: 6025.93, observed m / z: 1004.56 ([M / 6+H]) + ) and 861.48([M / 7+H + It was shown that a single main peak having ]) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by pre-HPLC (TFA conditions). BCY10570 (7.2 mg, 1.17 μmol, 53.90% yield, 97.95% purity) was obtained as a white solid.

[0411] BCY10574

[0412]

[0413] Preparation process of Compound 2

[0414]

[0415] DIEA (5.25 mg, 40.61 μmol, 7.07 μl, 1.5 eq) was added to a solution of BCY9594 (65 mg, 27.07 μmol, 1 eq) and Compound 1 (12.00 mg, 27.75 μmol, 1.02 eq) in DMSO (1 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS confirmed that BCY9594 was completely eliminated and the target m / z (calculated MW: 2718.13, observed m / z: 906.04([M / 3+H] + ), 1359.07([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide the residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (42.6 mg, 15.67 μmol, 57.89% yield, 100% purity) was obtained as a white solid.

[0416] Manufacturing process of BCY10574

[0417]

[0418] A mixture of compound 2 (20 mg, 7.36 μmol, 1.0 eq), BCY8927 (17 mg, 7.87 μmol, 1.07 eq), and THPTA (0.4 M, 18.4 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 18.4 μl, 1.0 eq) and VcNa (0.4 M, 36.8 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25–30°C for 12 hours under an N2 atmosphere. LC-MS confirmed that Compound 2 was completely extinguished and the target m / z (calculated MW: 4877.68, observed m / z: 1219.42 [M / 4+H]) was achieved. + ) and 975.54([M / 5+H + It was shown that a single main peak having ]) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY10574 (17.6 mg, 3.41 μmol, 46.29% yield, 94.40% purity) was obtained as a white solid.

[0419] BCY10575

[0420]

[0421] Preparation process of Compound 2

[0422]

[0423] DIEA (5.25 mg, 40.61 μmol, 7.07 μl, 1.5 eq) was added to a solution of BCY9594 (65 mg, 27.07 μmol, 1 eq) and Compound 1 (12.00 mg, 27.75 μmol, 1.02 eq) in DMSO (1 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS confirmed that Compound 1 was completely extinguished and the target m / z [Calculated MW: 2718.13 observed m / z : 906.04([M / 3+H] + ), and 1359.07([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide the residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (42.6 mg, 15.67 μmol, 57.89% yield, 100% purity) was obtained as a white solid.

[0424] BCY10575 manufacturing process

[0425]

[0426] A mixture of compound 2 (20 mg, 7.36 μmol, 1.0 eq), BCY8928 (17 mg, 7.67 μmol, 1.04 eq), and THPTA (0.4 M, 18.4 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 18.4 μl, 1.0 eq) and VcNa (0.4 M, 36.8 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25–30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 2 was completely extinguished and the target m / z [Calculated MW: 4935.71, Observed m / z: 1234.59 ([M / 4+H]] + ) and 987.71([M / 5+H + It was shown that a single main peak having ]) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY10575 (12 mg, 2.37 μmol, 32.27% yield, 97.67% purity) was obtained as a white solid.

[0427] BCY10576

[0428]

[0429] Preparation process of Compound 2

[0430]

[0431] DIEA (2.42 mg, 18.74 μmol, 3.3 μl, 1.5 eq) was added to a solution of BCY9594 (30.0 mg, 12.50 μmol, 1.0 eq) and Compound 1 (5.54 mg, 12.81 μmol, 1.02 eq) in DMSO (1 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS confirmed that Compound 1 was completely quenched and the target m / z [Calculated MW: 2718.3, Observed m / z: 906.45 ([M / 3+H]] + ) and 1359.50([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (16 mg, 5.80 μmol, 46.42% yield, 98.54% purity) was obtained as a white solid.

[0432] BCY10576 manufacturing process

[0433]

[0434] A mixture of compound 2 (17.0 mg, 6.25 μmol, 1.0 eq), BCY11014 (13.6 mg, 6.25 μmol, 1.0 eq), and THPTA (0.4 M, 1.8 μl, 2.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 15.6 μl, 1.0 eq) and VcNa (0.4 M, 1.84 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25–30°C for 12 hours under an N2 atmosphere. LC-MS showed that most of Compound 2 was completely extinguished and the target m / z [Calculated MW: 4893.63, Observed m / z: 1224.7 ([M / 4+H]] +) and 980.0([M / 6+H + It was shown that a single main peak having ]) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY10576 (20.5 mg, 4.13 μmol, 66.02% yield, 98.57% purity) was obtained as a white solid.

[0435] BCY10577

[0436]

[0437] Preparation process of Compound 2

[0438]

[0439] EDCI (8.5 mg, 54.8 μmol, 1.1 eq) and HOSu (5.7 mg, 49.5 μmol, 1.0 eq) were added to a solution of Compound 1 (5.0 mg, 49.5 μmol, 1.0 eq) in DMF (1 mL). The mixture was stirred at 25-30°C for 30 minutes. TLC indicated that Compound 1 had completely disappeared and a new spot had formed. Subsequently, BCY9172 (53 mg, 25.29 μmol, 0.47 eq) and DIEA (3.27 mg, 25.29 μmol, 4.4 μl, 0.47 eq) were added to the reaction mixture. The mixture was stirred at 25-30°C for 2 hours. LC-MS showed that BCY9172 had completely vanished and the target m / z (MW: 2178.46, observed m / z : 1089.5700([M / 2+H + It was shown that a single main peak having ]) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by pre-HPLC (neutral conditions). Compound 2 (30 mg, 13.77 μmol, 54.45% yield, 100% purity) was obtained as a white solid.

[0440] BCY10577 manufacturing process

[0441]

[0442] Compound 2 (20 mg, 9.18 μmol, 1.0 eq) and BCY6169 (32.95 mg, 10.10 μmol, 1.1 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 23 µl, 1 eq), VcNa (0.4 M, 46 µl, 2.0 eq), and THPTA (0.4 M, 23 µl, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 4 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 5441.20, observed m / z: 1361.8 ([M / 4+H]) + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by pre-HPLC (TFA conditions). BCY10577 (16.2 mg, 2.98 μmol, 32.43% yield) was obtained as a white solid.

[0443] Example 3: Synthesis of Nectin-4 / CD137 Binded Heterotandem Acyclic Peptide

[0444] BCY8854

[0445]

[0446] General manufacturing process of BCY8846

[0447]

[0448] DIEA (52.5 mg, 406 μmol, 70.8 μl, 4.0 eq) was added to a solution of BCY8234 (a peptide identical to BCY8846 except for the absence of the PYA moiety; 300 mg, 102 μmol, 1.0 eq) in DMA (3 mL) while stirring for 10 minutes. Subsequently, (2,5-dioxopyrrolidine-1-yl)pent-4-inoate (25.8 mg, 132 μmol, 1.3 eq) was added to the mixture, and the mixture was stirred for an additional 16 hours at 20°C. LC-MS confirmed that BCY8234 was completely eliminated and the target m / z (calculated MW: 3034.43, observed m / z : 1011.8([M / 3+H] + ), 1517.0([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (neutral conditions) to provide compound BCY8846 (290 mg, 95.6 μmol, 94.1% yield) as a white solid.

[0449] General manufacturing process of BCY8854

[0450]

[0451] BCY7859 (which can be prepared as described in BCY7985; 220 mg, 77.8 μmol, 1.0 eq) was added to a solution of BCY8846 (234 mg, 77.1 μmol, 1.0 eq) in DMF (5 mL), followed by the addition of (2R)-2[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (0.80 M, 963 μl, 1.0 eq) and CuSO4 (0.80 M, 289 μl, 0.3 eq). The mixture was stirred at 20°C for 2 hours. LC-MS confirmed that BCY8846 was completely eliminated and the target m / z (calculated MW: 5861.59, observed m / z : 837.9([M / 7+H] + ), 977.6([M / 6+H] + ), 1173.3([M / 5+H]+ It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (A: 0.075% TFA in H2O, B: ACN) to provide compound BCY8854 (292 mg, 46.8 μmol, 60.8% yield, 95.9% purity, TFA) as a white solid.

[0452] BCY9350

[0453]

[0454] General manufacturing process of BCY8782-PYA

[0455]

[0456] DIEA (4.37 mg, 33.9 μmol, 5.90 μl, 5.0 eq) and (2,5-dioxopyrrolidine-1-yl)pent-4-inoate (2.64 mg, 13.5 μmol, 2.0 eq) were added to a solution of BCY8782 (a peptide identical to BCY11942 except for the absence of the PYA moiety; 20.0 mg, 6.77 μmol, 1.0 eq) in DMA (1 mL) while stirring at 25°C for 12 hours. LC-MS confirmed that BCY8782 was completely eliminated and the target m / z (calculated MW: 3034.43, observed m / z : 1012.1([M / 3+H] + It was shown that a single main peak having ) was detected. The reaction mixture was purified by pre-HPLC (neutral conditions) to provide compound BCY11942 (20.0 mg, 6.00 μmol, 88.6% yield, 91.0% purity) as a white solid.

[0457] General manufacturing process of BCY9350

[0458]

[0459] (2R)-2[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxy-2H-furan-5-one (0.4M, 330 µl, 20.0 eq) was added to a solution of BCY11942 (20 mg, 6.59 μmol, 1.0 eq) and BCY7859 (can be prepared as described in BCY7985; 20.5 mg, 7.25 μmol, 1.1 eq) in DMF (1 mL), and CuSO4 (0.4M, 98.9 µl, 6.0 eq) was added to the mixture. The mixture was stirred at 25°C for 2 hours. LC-MS confirmed that BCY8782-PYA was completely eliminated and the target m / z (calculated MW: 5861.59, observed m / z : 1173.3([M / 5+H] + It was shown that a single main peak having ) was detected. The reaction mixture was purified by pre-HPLC (A: 0.075% TFA in H2O, B: ACN) to provide BCY9350 (14.5 mg, 2.40 μmol, 36.5% yield, 97.2% purity) as a white solid.

[0460] BCY9351

[0461]

[0462] General manufacturing process of BCY9351

[0463]

[0464] Vc (0.4 M, 165 µl, 20.0 eq) and CuSO4 (0.4 M, 49.4 µl, 6.0 eq) were added under a nitrogen atmosphere to a solution of BCY8940 (can be prepared as described in BCY8942; 9.4 mg, 3.33 μmol, 1.01 eq) and BCY8846 (10.0 mg, 3.30 μmol, 1.0 eq) in DMF (1 mL). The mixture was stirred at 25°C for 1 hour. LC-MS confirmed that BCY8940 was completely eliminated and the target m / z (calculated MW: 5861.59, observed m / z : 975.4[M / 6+H] +, 1172.3([M / 5+H] + It was shown that a single main peak having ) was detected. The reaction mixture was purified by pre-HPLC (A: 0.075% TFA in H2O, B: ACN) to provide BCY9351 (5.30 mg, 0.904 μmol, 26.3% yield, 96.0% purity) as a white solid.

[0465] BCY9399

[0466]

[0467] Preparation process of Compound 2

[0468]

[0469] TEA (8.65 mg, 85.45 μl, 11.9 eq) was added to a solution of COM134 (30 mg, 56.97 μmol) and Compound 1 (17.22 mg, 85.45 μmol) in DCM (0.5 mL). The mixture was stirred at 25°C for 1 hour. LC-MS confirmed that COM134 was completely eliminated and the target m / z (calculated MW: 691.72, observed m / z : 692.3([M+H] + ) and 709.3([M+NH4] + It was shown that a single main peak having ) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (neutral conditions). Compound 2 (30.5 mg) was obtained as a colorless oil.

[0470] Preparation process of Compound 3

[0471]

[0472] DIEA (8.41 mg, 65.05 μmol, 11.33 μl) was added to a solution of Compound 2 (15 mg, 21.68 μmol) and BCY8116 (47 mg, 21.68 μmol) in DMF (1 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (calculated MW: 2725.1 observed) m / z : 1362.7([M / 2+H] + ), 909.0([M / 3+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (20 mg, 33.41% yield, 98.71% purity) was obtained as a white solid.

[0473] BCY9399 manufacturing process

[0474]

[0475] A mixture of compound 3 (20.0 mg, 5.35 μmol, 1.0 eq), BCY7741 (13.0 mg, 5.70 μmol, 1.01 eq), and THPTA (0.4 M, 13.4 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 13.4 μl, 1.0 eq) and VcNa (0.4 M, 26.8 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 5006.64] was observed. m / z : 834.9([M / 6+H] + ), 1002.3([M / 5+H] + ), 1252.4([M / 4+H]+ It was shown that a single main peak having ) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9399 (9.1 mg, 27.20% yield, 96.29% purity) was obtained as a white solid.

[0476] BCY9400

[0477]

[0478] Preparation process of Compound 2

[0479]

[0480] TEA (4.14 mg, 40.94 μL, 5.7 eq) was added to a solution of COM135 (can be prepared as described in BCY9648; 30.0 mg, 27.29 μmol) and Compound 1 (8.3 mg, 40.94 μmol) in DCM (2 mL). The reaction mixture was then stirred at 25-30°C for 1 hour. LC-MS confirmed that COM135 was completely eliminated and the target m / z (calculated MW: 1264.40, observed m / z : 1281.4([M+NH4] + It was shown that a single main peak having ) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (neutral conditions) to provide compound 2 (18 mg) as a white solid.

[0481] Preparation process of Compound 3

[0482]

[0483] DIEA (1.4 mg, 10.68 μmol, 1.9 μl) was added to a solution of Compound 2 (15.5 mg, 7.12 μmol) and BCY8116 (9 mg, 7.12 μmol) in DMF (2 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 3297.78, observed m / z: 1099.7([M / 3+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (19.5 mg, 5.91 μmol, 33.41% yield, 83.07% purity) was obtained as a white solid.

[0484] BCY9400 manufacturing process

[0485]

[0486] A mixture of Compound 3 (19.5 mg, 5.91 μmol), BCY7741 (14 mg, 6.14 μmol, 1.01 eq), and THPTA (0.4 M, 15 μl, 1 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 15 μl, 1 eq) and VcNa (0.4 M, 30 μl, 2 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred under an N2 atmosphere at 25-30°C for 12 hours. LC-MS showed that compound 3 was completely extinguished and the target m / z [MW: 5579.31] was observed m / z : 930.5([M / 6+H] + ), 1116.6([M / 5+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9400 (13.9 mg, 2.33 μmol, 27.20% yield, 93.56% purity) was obtained as a white solid.

[0487] BCY9401

[0488]

[0489] Preparation process of Compound 3

[0490]

[0491] TEA (4.8 mg, 47.09 μmol, 6.6 μl, 1.5 eq) was added to a solution of Compound 1 (50.0 mg, 31.39 μmol, 1 eq) and Compound 2 (6.6 mg, 32.96 μmol, 1.05 eq) in DCM (2 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS indicated that Compound 1 was completely eliminated and the target m / z (MW: 1757.86) was observed. m / z : 879.10([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 3 (0.02 g, 6.56 μmol, 20.91% yield, 57.7% purity) was obtained as a white solid.

[0492] Preparation process of Compound 4

[0493]

[0494] DIEA (2.2 mg, 17.07 μmol, 2.97 μl, 1.5 eq) was added to a solution of Compound 3 (20 mg, 11.38 μmol, 1 eq) and BCY8116 (25 mg, 11.51 μmol, 1.01 eq) in DMF (4 mL). The mixture was stirred at 25-30°C for 12 hours. LC-MS confirmed that Compound 3 was completely eliminated and the target m / z (MW: 3791.23, observed m / z : 1263.2([M / 3+H] + It was shown that a single main peak having )) was detected. The reaction product was directly purified by pre-HPLC (neutral conditions). Compound 4 (10 mg, 2.43 μmol, 21.33% yield, 92% purity) was obtained as a colorless oil.

[0495] BCY9401 manufacturing process

[0496]

[0497] A mixture of compound 4 (10 mg, 2.43 μmol, 0.9 eq), BCY7741 (6.32 mg, 2.77 μmol, 1.0 eq), and THPTA (0.4 M, 6.7 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 6.7 μl, 1.0 eq) and VcNa (0.4 M, 13.4 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS confirmed that Compound 4 was completely extinguished and the target m / z [MW: 6072.77, observed m / z : 1012.00([M / 6+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9401 (8.4 mg, 1.56 μmol, 59.31% yield, 95.52% purity) was obtained as a white solid.

[0498] BCY9403

[0499]

[0500] Preparation process of Compound 2

[0501]

[0502] TEA (11.6 mg, 114.64 μmol, 16.0 μl, 1.5 eq) was added to a solution of COM471 (100.0 mg, 76.42 μmol, 1.0 eq) and 4-nitrophenylchloroformate (16.2 mg, 80.25 μmol, 1.05 eq) in DCM (10 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS confirmed that COM471 was completely eliminated and the target m / z (MW: 1473.58, observed m / z : 736.83([M / 2+H] + It was shown that a single main peak having ) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (62.8 mg, 42.67 μmol, 55.84% yield, 48.37% purity) was obtained as a white oil.

[0503] Preparation process of Compound 3

[0504]

[0505] DIEA (5.66 mg, 43.77 μmol, 7.62 μl, 1.5 eq) was added to a solution of Compound 2 (44 mg, 29.46 μmol, 1.0 eq) and BCY8116 (63 mg, 29.18 μmol, 1.0 eq) in DMF (2 mL). The mixture was stirred at 40°C for 12 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 3506.95, observed m / z : 1168.58([M / 3+H] + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (TFA conditions). Compound 3 (20 mg, 5.42 μmol, 18.57% yield, 95.04% purity) was obtained as a white solid.

[0506] BCY9403 manufacturing process

[0507]

[0508] A mixture of Compound 3 (10.0 mg, 2.71 μmol, 1.0 eq), BCY7741 (6.83 mg, 2.99 μmol, 1.1 eq), and THPTA (0.4 M, 7 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 7 μl, 1 eq) and VcNa (0.4 M, 14 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred under an N2 atmosphere at 25-30°C for 12 hours. LC-MS showed that compound 3 was completely extinguished and the target m / z [MW: 5788.49, observed m / z : 1157.00([M / 5+H] + ) and 964.60([M / 6+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9403 (2.1 mg, 0.34 μmol, 11.93% yield, 93.80% purity) was obtained as a white solid.

[0509] BCY9405

[0510]

[0511] Preparation process of Compound 2

[0512]

[0513] TEA (5.8 mg, 57.14 μmol, 8 μl) was added to a solution of COM472 (44.7 mg, 38.1 μmol) and Compound 1 (9.2 mg, 45.72 μmol) in DCM (4 mL). The mixture was stirred at 25°C for 2 hours. LC-MS confirmed that COM472 was completely eliminated and the target m / z (MW: 1338.45, observed m / z : 686.23([M / 2+NH4+ It was shown that a single main peak having ]) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (neutral conditions). Compound 2 (20 mg, 14.94 μmol, 39.2% yield) was obtained as a colorless oil.

[0514] Preparation process of Compound 3

[0515]

[0516] DIEA (1.9 mg, 14.94 μmol, 2.6 μl) was added to a solution of Compound 2 (20 mg, 14.94 μmol) and BCY8116 (38.96 mg, 17.93 μmol) in DMF (4 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 3371.82, observed m / z : 1123.94([M / 3+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (10 mg, 99.07% yield, 19.66 purity) was obtained as a white solid.

[0517] BCY9405 manufacturing process

[0518]

[0519] A mixture of compound 3 (10.0 mg, 2.97 μmol, 1.0 eq), BCY7741 (7.4 mg, 3.26 μmol, 1.1 eq), and THPTA (1.3 mg, 2.97 μmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 7.5 μL, 1.0 eq) and VcNa (0.4 M, 151 μL, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by titrating 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 5653.36] was observed. m / z : 1130.47([M / 5+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9405 (7.8 mg, 46.08% yield, 97.8% purity) was obtained as a white solid.

[0520] BCY9406

[0521]

[0522] Preparation process of Compound 2

[0523]

[0524] TEA (27.0 mg, 266.09 μmol, 37 μL, 1.5 eq) was added to a solution of COM473 (130.0 mg, 177.40 μmol, 1.0 eq) and (4-nitrophenyl)carbonochloridate (36.4 mg, 180.59 μmol, 1.02 eq) in DCM (3 mL). The mixture was stirred at 35°C for 2 hours. LC-MS confirmed that COM473 was completely eliminated and the target m / z (MW: 897.93, observed m / z : 897.65([M+H]+ ), 914.60([M+NH + It was shown that a single main peak having ])) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (90 mg, 95.87 μmol, 54.04% yield, 95.65% purity) was obtained as a colorless oil.

[0525] Preparation process of Compound 3

[0526]

[0527] DIEA (2.16 mg, 16.71 μmol, 2.91 μl, 1.5 eq) was added to a solution of Compound 2 (10 mg, 11.14 μmol, 1 eq) and BCY8116 (25 mg, 11.51 μmol, 1.03 eq) in DMF (2 mL). The mixture was stirred at 25-30°C for 12 hours. LC-MS was performed at the target m / z (MW: 2931.30, observed m / z : 977.00([M / 3+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (FTA conditions). Compound 3 (15 mg, 5.12 μmol, 45.79% yield, 99.66% purity) was obtained as a white solid.

[0528] BCY9406 manufacturing process

[0529]

[0530] A mixture of compound 3 (15 mg, 5.12 μmol, 1.0 eq), BCY7741 (12 mg, 5.26 μmol, 1.03 eq), and THPTA (0.4 M, 12.8 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 12.8 μl, 1.0 eq) and VcNa (0.4 M, 25.6 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 5212.84] was observed. m / z : 1042.74([M / 4+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9406 (14.4 mg, 2.57 μmol, 50.21% yield, 93.01% purity) was obtained as a white solid.

[0531] BCY9407

[0532]

[0533] Preparation process of Compound 2

[0534]

[0535] The solution of COM128 (60 mg, 50.53 μmol, 1.0 eq), Compound 1 (13 mg, 64.50 μmol, 1.28 eq), and DIEA (9.80 mg, 75.80 μmol, 13.20 μl, 1.5 eq) in DCM (5 mL) was degassed and purged with N2 three times, followed by stirring the mixture under an N2 atmosphere at 25-30°C for 1 hour. LC-MS confirmed that COM128 was completely eliminated and the target m / z (calculated MW: 1352.48, observed m / z : 676.7([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (12 mg, 8.87 μmol, 17.56% yield) was obtained as a colorless oil.

[0536] Manufacturing process of [BCY8116]-[COM128]

[0537]

[0538] The solution of Compound 2 (7 mg, 5.18 μmol, 1.0 eq), BCY8116 (11 mg, 5.06 μmol, 1.0 eq), and DIEA (2.01 mg, 15.53 μmol, 2.70 μl, 3.0 eq) in DMF (3 mL) was degassed and purged with N2 three times, followed by stirring the mixture under an N2 atmosphere at 25-30°C for 1 hour. LC-MS was performed at the target m / z (calculated MW: 3385.85, observed m / z : 1129.3([M / 3+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). [BCY8116]-[COM128] (15.6 mg, 4.46 μmol, 86.13% yield, 96.75% purity) was obtained as a white solid.

[0539] BCY9407 manufacturing process

[0540]

[0541] A mixture of [BCY8116]-[COM128] (15.6 mg, 4.61 μmol, 1.0 eq), BCY7741 (11 mg, 4.82 μmol, 1.05 eq), and THPTA (0.8 M, 5.8 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 11.6 μl, 1.0 eq) and VcNa (0.4 M, 23.2 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25–30°C for 12 hours under an N2 atmosphere. LC-MS was used to determine the target m / z (calculated MW: 5667.39, observed m / z : 945.6([M / 6+H] + ) and 1134.2)[M / 5+H] + It was shown that a single main peak having )) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9407 (1.3 mg, 0.23 μmol, 4.33% yield, 86.90% purity) was obtained as a white solid.

[0542] BCY9408

[0543]

[0544] Preparation process of Compound 2

[0545]

[0546] TEA (5.5 mg, 53.88 μmol, 7.5 μL, 1.5 eq) was added to a solution of COM129 (45.0 mg, 34.39 μmol, 1.0 eq) and Compound 1 (15.0 mg, 74.42 μmol, 2.1 eq) in DCM (5 mL), and the mixture was then stirred under an N2 atmosphere at 25-30°C for 1 hour. LC-MS confirmed that COM129 was completely eliminated and the target m / z (MW: 1473.58, observed m / z : 737.3([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (9 mg, 6.11 μmol, 17.01% yield, 95.76% purity) was obtained as a white solid.

[0547] Preparation process of Compound 3

[0548]

[0549] DIEA (2.4 mg, 18.32 μmol, 3.2 μl, 3.0 eq) was added to a solution of Compound 2 (9.0 mg, 6.11 μmol, 1.0 eq) and BCY8116 (13.3 mg, 6.11 μmol, 1.0 eq) in DMF (3 mL). All solvents were degassed and purged with N2 three times, followed by stirring the mixture under an N2 atmosphere at 25-30°C for 1 hour. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 3506.95, observed m / z : 877.4([M / 4+H] + ) and m / z: 1169.6([M / 3+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (7.2 mg, 2.05 μmol, 31.93% yield, 95% purity) was obtained as a white solid.

[0550] BCY9408 manufacturing process

[0551]

[0552] A mixture of compound 3 (7.2 mg, 2.05 μmol, 1.0 eq), BCY7741 (5.0 mg, 2.19 μmol, 1.03 eq), and THPTA (0.4 M, 5.1 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 5.1 μl, 1.0 eq) and VcNa (0.4 M, 10.2 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 5788.49] was observed. m / z : 968.9([M / 6+H] + ) and 1158.0([M / 5+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9408 (3.1 mg, 4.97e-1 μmol, 24.23% yield, 92.87% purity) was obtained as a white solid.

[0553] BCY9409

[0554]

[0555] Preparation process of Compound 2

[0556]

[0557] TEA (3.15 mg, 31.17 μmol, 4.34 μl, 1.5 eq) was added to a solution of Compound 1 (30.0 mg, 20.78 μmol) and COM130 (6.28 mg, 31.17 μmol) in DCM (3 mL). The mixture was stirred at 25-30°C for 1 hour. LC-MS confirmed that Compound 1 was completely eliminated and the target m / z (MW: 1608.70) was observed. m / z: 804.8([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure and then freeze-dried to provide compound 2 (10.2 mg, crude material) as a white solid.

[0558] Preparation process of Compound 3

[0559]

[0560] DIEA (0.8 mg, 6.22 μmol, 1.1 μL, 1.0 eq) was added to a solution of Compound 2 (10.2 mg, 6.34 μmol) and BCY8116 (13.50 mg, 6.22 μmol) in DMF (2 mL). The mixture was stirred at 30°C for 2 hours. LC-MS was performed at the target m / z (MW: 3642.08, observed m / z : 1214.4([M / 3+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (15.0 mg, 4.12 μmol, 62.94% yield, 95% purity) was obtained as a white solid.

[0561] BCY9409 manufacturing process

[0562]

[0563] A mixture of compound 3 (15 mg, 4.12 μmol, 1.0 eq), BCY7741 (10 mg, 4.38 μmol, 1.03 eq), and THPTA (0.4 M, 10.3 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 10.3 μl, 1.0 eq) and VcNa (0.4 M, 20.6 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 5923.61, observed m / z : 988.2([M / 4+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9409 (3.1 mg, 0.52 μmol, 12.62% yield, 90.89% purity) was obtained as a white solid.

[0564] BCY9410

[0565]

[0566] Preparation process of Compound 2

[0567]

[0568] TEA (36.4 mg, 359.23 μmol, 50.0 μl, 1.6 eq) was added to a solution of COM131 (167.0 mg, 227.89 μmol, 1.0 eq) and Compound 1 (55.0 mg, 272.87 μmol, 1.2 eq) in DCM (5 mL). The mixture was stirred at 25-30°C for 1 hour. LC-MS was performed at the target m / z (MW: 897.93) and the observed value was 920.3 ([M+Na +It was shown that a single main peak having ])) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (35 mg, 33.74 μmol, 14.81% yield, 86.56% purity) was obtained as a colorless oil.

[0569] Preparation process of Compound 3

[0570]

[0571] DIEA (8.64 mg, 66.82 μmol, 11.64 μl, 3.0 eq) was added to a solution of Compound 2 (20 mg, 22.27 μmol, 1.0 eq) and BCY8116 (48 mg, 22.09 μmol, 1.0 eq) in DMF (2 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 3 was completely eliminated and the target m / z (MW: 2931.32, observed m / z : 977.7([M+H] + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (TFA conditions). Compound 3 (40 mg, 13.08 μmol, 58.7% yield, 95.82% purity) was obtained as a white solid.

[0572] BCY9410 manufacturing process

[0573]

[0574] A mixture of Compound 3 (40 mg, 13.08 μmol, 1.0 eq), BCY7741 (35 mg, 15.34 μmol, 1.17 eq), and THPTA (0.4 M, 34 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 34 μl, 1.0 eq) and VcNa (0.4 M, 68 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred under an N2 atmosphere at 25-30°C for 12 hours. LC-MS showed that compound 3 was completely extinguished and the target m / z [MW: 5212.85, observed m / z : 1043.2([M / 5+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9410 (38.6 mg, 6.78 μmol, 49.71% yield, 91.6% purity) was obtained as a white solid.

[0575] BCY9411

[0576]

[0577] Preparation process of Compound 2

[0578]

[0579] TEA (2.8 mg, 24.48 μmol, 3.4 μl, 1.5 eq) was added to a solution of COM132 (5 mg, 16.32 μmol, 1 eq) and Compound 1 (4 mg, 19.85 μmol, 1.22 eq) in DCM (5 mL). The mixture was stirred at 25°C for 1 hour. LC-MS was performed at the target m / z (calculated MW: 471.46, observed m / z : 489.2([M+NH4] +It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent, and then freeze-dried to provide compound 2 (8 mg, crude material) as a white solid.

[0580] Preparation process of Compound 3

[0581]

[0582] DIEA (0.7 mg, 6.90 μmol, 1 μL, 1.5 eq) was added to a solution of Compound 2 (3.3 mg, 6.9 μmol, 1.5 eq) and BCY8116 (10.0 mg, 4.6 μmol, 1.0 eq) in DMF (5 mL). The mixture was stirred at 30°C for 2 hours. LC-MS confirmed that Compound 2 was completely extinguished and the target m / z (calculated MW: 2504.83, observed m / z : 1252.3([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). Compound 3 (4.2 mg, 1.51 μmol, 32.78% yield, 90% purity) was obtained as a white solid.

[0583] BCY9411 manufacturing process

[0584]

[0585] A mixture of compound 3 (4.2 mg, 1.68 μmol, 1.0 eq), BCY7741 (4.0 mg, 1.75 μmol, 1.05 eq), and THPTA (0.04 M, 84 μl, 2.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.04 M, 84 μl, 2.0 eq) and VcNa (0.04 M, 168 μl, 4.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z [MW: 4786.37] was observed. m / z : 1596.2([M / 3+H] + ), 1196.9([M / 4+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY9411 (4.1 mg, 0.86 μmol, 50.20% yield, 98.26% purity) was obtained as a white solid.

[0586] BCY9759

[0587]

[0588] Preparation process of Compound 2

[0589]

[0590] DIEA (0.9 mg, 7.07 μmol, 1.2 μL, 2.0 eq) was added to a solution of Compound 1 (5.0 mg, 3.54 μmol, 1.0 eq) and BCY8116 (7.7 mg, 3.54 μmol, 1.0 eq) in DCM (3 mL). The mixture was stirred at 0°C for 20 minutes. LC-MS detected the mass corresponding to Compound 2 after the NHS group was removed (calculated MW: 3470.95, hydrolyzed MW: 3373.81, observed m / z : 1125.0([M / 3+H] + The reaction mixture was filtered, concentrated under reduced pressure, and freeze-dried to obtain compound 2 (15 mg, crude material) as a white solid.

[0591] BCY9759 manufacturing process

[0592]

[0593] DIEA (1.5 mg, 11.52 μmol, 2.0 μL, 2.0 eq) was added to a solution of Compound 2 (20 mg, 5.76 μmol, 1.0 eq) and BCY7732 (12.7 mg, 5.76 μmol, 1.0 eq) in DMF (3 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS confirmed that Compound 2 was completely eliminated and the target m / z (MW: 5557.3, observed m / z : 927.0([M / 6+H] + ) and 1112.2([M / 5+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by reverse-phase HPLC (TFA conditions). BCY9759 (2.3 mg, 6.92% yield, 96.29% purity) was obtained as a white solid.

[0594] BCY10000

[0595]

[0596] Manufacturing process of BCY9172-PEG12-N3

[0597]

[0598] BCY9172 (520 mg, 248.16 μmol, 1.0 eq) and Compound 1 (370 mg, 499.47 μmol, 2.01 eq) were dissolved in DMF (5 mL), DIEA (48.11 mg, 372.24 μmol, 64.84 μl, 1.5 eq) was added to the mixture, and the mixture was stirred at 30°C for 12 hours. LC-MS confirmed that BCY9172 was completely eliminated and the target m / z (calculated MW: 2721.12, observed m / z: 1360.9 [M / 2+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (284 mg, 101.10 μmol, 40.74% yield, 96.87% purity) was obtained as a white solid.

[0599] BCY10000 manufacturing process

[0600]

[0601] The reaction was carried out in parallel in two independent vessels. For one vessel, Compound 2 (142 mg, 52.18 μmol, 1.0 eq) and BCY8846 (157 mg, 51.74 μmol, 1.0 eq) were first dissolved in 10 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 130.5 μL, 1.0 eq), VcNa (0.4 M, 261.0 μL, 2.0 eq), and THPTA (0.4 M, 130.5 μL, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 12 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 5755.54, observed m / z: 959.60 ([M / 6+H])+ ) and 1151.55([M / 5+H] + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY10000 (314.9 mg, 51.99 μmol, 49.82% yield, 95.03% purity) was obtained as a white solid.

[0602] BCY10567

[0603]

[0604] Manufacturing process of BCY8919-PEG12-N3

[0605]

[0606] BCY8919 (60.0 mg, 28.85 μmol, 1.0 eq) and Compound 1 (22.2 mg, 30.01 μmol, 1.04 eq) were dissolved in DMSO (1 mL). DIPEA (5.6 mg, 43.28 μmol, 7.6 μl, 1.5 eq) was added to the solution, and the mixture was then stirred at 25–30°C for 2 hours. LC-MS confirmed that BCY8919 was completely eliminated and the target m / z (calculated MW: 2705.16, observed m / z: 1353.15 [M / 2+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (BCY8919-PEG12-N3, 18.5 mg, 6.77 μmol, 23.47% yield, 99.04% purity) was obtained as a white solid.

[0607] BCY10567 manufacturing process

[0608]

[0609] Note: This reaction was performed twice, and the first reaction is described below.

[0610] Compound 2 (9.0 mg, 3.33 μmol, 1.0 eq) and BCY8846 (10.1 mg, 3.33 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 8.3 μl, 1.0 eq), VcN (1.3 mg, 6.56 μmol, 2.0 eq), and THPTA (1.4 mg, 3.22 μmol, 1.0 eq). Finally, 0.4 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 16 hours under an N2 atmosphere. LC-MS showed that Compound 2 was completely extinguished and the target m / z (calculated MW: 5739.58, observed m / z: 956.75 ([M / 6+H]) + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY10567 (6.85 mg, 1.18 μmol, 35.48% yield, 98.91% purity) was obtained as a white solid.

[0611] BCY10569

[0612]

[0613] Preparation process of Compound 3

[0614]

[0615] A mixture of compound BCY8920 (40.0 mg, 18.71 μmol, 1.0 eq), compound 2 (16.0 mg, 21.6 μmol, 1.15 eq), and DIEA (5.0 μl, 28.0 μmol, 1.5 eq) was dissolved in DMF. The reaction mixture was evaluated at 40°C for 1 hour, and LC-MS was performed at the target m / z (calculated MW: 2763.2, observed m / z : 912.17([(M-28) / 2+H] +The mixture was stirred until it was shown that a single main peak having ) was detected. The reaction mixture was then concentrated under reduced pressure to remove the solvent and produce a residue, which was then purified by pre-HPLC (under TFA conditions). Compound 3 (23.4 mg, 8.47 μmol, 45.25% yield, 99.0% purity) was obtained as a white solid.

[0616] BCY10569 manufacturing process

[0617]

[0618] A mixture of Compound 3 (5.0 mg, 1.81 μmol, 1.0 eq), BCY8846 (5.8 mg, 1.9 μmol, 1.05 eq), and THPTA (1.0 mg, 2.3 μmol, 1.3 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 5.0 μl, 1.0 eq) and VcNa (0.4 M, 5.0 μl, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS showed that compound 3 was completely extinguished and the target m / z (calculated MW: 5797.62, observed m / z : 1160.7([M / 5+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was directly purified by pre-HPLC (TFA conditions) and BCY10569 (5.7 mg, 1.18 μmol, 52.25% yield, 96.16% purity) was obtained as a white solid.

[0619] BCY10571

[0620]

[0621] Manufacturing process of BCY8116-PEG5-N3

[0622]

[0623] BCY8116 (60 mg, 27.62 μmol, 1.0 eq) and Compound 1 (12.0 mg, 27.75 μmol, 1.0 eq) were first dissolved in DMSO (1 mL), and then DIEA (5.4 mg, 41.43 μmol, 7.22 μl, 1.5 eq) was added to the mixture. The mixture was stirred at 30°C for 12 hours. LC-MS was performed at the target m / z (MW: 2489.82, observed m / z: 1245.1700 [M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (48 mg, 19.28 μmol, 69.80% yield, 100% purity) was obtained as a white solid.

[0624] BCY10571 manufacturing process

[0625]

[0626] The reaction was carried out in parallel in two independent vessels. For one vessel, Compound 2 (10 mg, 4.02 μmol, 1.0 eq) and BCY8927 (9 mg, 4.17 μmol, 1.04 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 10.0 µl, 1.0 eq), VcNa (0.4 M, 20.1 µl, 2.0 eq), and THPTA (0.4 M, 10.0 µl, 1 eq). Finally, 0.4 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 4 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and at the target m / z (MW: 4649.36, observed m / z: 1162.57 ([M / 4+H]) + ), 1549.69([M / 3+H] + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (TFA conditions). BCY10571 (13 mg, 2.79 μmol, 34.88% yield, 96.48% purity) was obtained as a white solid.

[0627] BCY10572

[0628]

[0629] Manufacturing process of BCY8116-PEG5-N3

[0630]

[0631] BCY8116 (60 mg, 27.62 μmol, 1.0 eq) and Compound 1 (12.0 mg, 27.75 μmol, 1.0 eq) were first dissolved in DMSO (1 mL), and then DIEA (5.4 mg, 41.43 μmol, 7.22 μl, 1.5 eq) was added to the mixture. The mixture was stirred at 30°C for 12 hours. LC-MS was performed at the target m / z (MW: 2489.82, observed m / z: 1245.1700 [M / 2+H]+ It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (48 mg, 19.28 μmol, 69.80% yield, 100% purity) was obtained as a white solid.

[0632] Manufacturing process of BCY10572

[0633]

[0634] The reaction was carried out in parallel in two independent vessels. For one vessel, Compound 2 (10 mg, 4.02 μmol, 1.0 eq) and BCY8928 (9 mg, 4.06 μmol, 1.01 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 10.1 µl, 1 eq), VcNa (0.4 M, 20.2 µl, 2.0 eq), and THPTA (0.4 M, 10.1 µl, 1.0 eq). Finally, 0.4 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 4 hours under an N2 atmosphere. LC-MS showed that Compound 1 was completely annihilated and at the target m / z (MW: 4707.40, observed m / z: 1568.29 ([M / 3+H]) + ) and 1176.83([M / 4+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). BCY10572 (21 mg, 4.46 μmol, 55.7% yield, 97.51% purity) was obtained as a white solid.

[0635] BCY10573

[0636]

[0637] Preparation process of Compound 2

[0638]

[0639] DIEA (3.12 mg, 24.17 μmol, 4.21 μl, 1.5 eq) was added to a solution of BCY8116 (35 mg, 16.11 μmol, 1 eq) and Compound 1 (7.00 mg, 16.19 μmol, 1 eq) in DMSO (1 mL). The mixture was stirred at 25-30°C for 2 hours. LC-MS indicated that most of the BCY8116 had been eliminated and the target m / z (calculated MW: 2489.82, observed 1245.37 [M / 2+H]) was achieved. + ) and 830.25([M / 3+H] + It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and provide a residue. The residue was purified by pre-HPLC (TFA conditions). Compound 2 (26.8 mg, 10.76 μmol, 66.81% yield, 100% purity) was obtained as a white solid.

[0640] BCY10573 manufacturing process

[0641]

[0642] A mixture of compound 2 (15 mg, 6.02 μmol, 1.0 eq), BCY11014 (13.50 mg, 6.21 μmol, 1.03 eq), and THPTA (0.4 M, 15.1 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 15.1 μl, 1.0 eq) and VcNa (0.4 M, 30.2 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25-30°C for 12 hours under an N2 atmosphere. LC-MS showed that Compound 2 was completely extinguished and the target m / z [MW: 4665.32, observed m / z : 1167.50([M / 4+H] + It was shown that a single main peak having )] was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY10573 (11.5 mg, 2.42 μmol, 40.14% yield, 98.11% purity) was obtained as a white solid.

[0643] BCY10578

[0644]

[0645] Preparation process of Compound 2

[0646]

[0647] Compound 1 (5.0 mg, 49.5 μmol, 1.0 eq) was first activated by mixing EDCI (8.5 mg, 54.8 μmol, 1.1 eq) and HOSu (5.7 mg, 49.5 μmol, 1.0 eq). The mixture was stirred at 25–30°C for 30 minutes. TLC indicated that Compound 1 was completely extinguished and a single new spot was formed. Subsequently, Compound BCY9172 (80.0 mg, 38.18 μmol, 0.8 eq) and DIEA (6.3 mg, 8.5 μl, 49.5 μmol, 1.0 eq) were added to the mixture, and LC-MS was performed at the target m / z (calculated MW: 2178.46, observed m / z: 1089.44 [M / 2+H]). + The mixture was stirred at 40°C for 1 hour until a single main peak having ) was detected. The reaction mixture was then concentrated under reduced pressure to remove the solvent and produce a residue, which was then purified by pre-HPLC (under TFA conditions). Compound 2 (15 mg, 6.88 μmol, 18.66% yield, 73.3% purity) was obtained as a white solid.

[0648] Manufacturing process of BCY10578

[0649]

[0650] A mixture of Compound 2 (9.8 mg, 4.5 μmol, 1.0 eq), BCY8846 (14.0 mg, 4.6 μmol, 1.0 eq), and THPTA (2.0 mg, 4.6 μmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 12 μL, 1.0 eq) and VcNa (0.4 M, 24 μL, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 5212.88, observed m / z : 1304.2([M / 4+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was directly purified by pre-HPLC (TFA conditions), and BCY10578 (13.78 mg, 2.64 μmol, 58.66% yield, 96.23% purity) was obtained as a white solid.

[0651] BCY10917

[0652]

[0653] Manufacturing process of BCY8831-PEG12-N3

[0654]

[0655] BCY8831 (40.0 mg, 13.29 μmol, 1.0 eq) and Compound 1 (10.5 mg, 14.17 μmol, 1.07 eq) were dissolved in DMF (1 mL). DIPEA (2.6 mg, 20.09 μmol, 3.5 μl, 1.5 eq) was added to the solution, and the mixture was then stirred at 30°C for 16 hours. LC-MS confirmed that BCY8831 was completely eliminated and the target m / z (calculated MW: 3635.16, observed m / z: 1212.0 [M / 3+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (22.0 mg, 5.83 μmol, 43.85% yield, 96.39% purity) was obtained as a white solid.

[0656] BCY10917 manufacturing process

[0657]

[0658] Note: Two batches were manufactured, and the first one was recorded for the final report.

[0659] Compound 2 (10.0 mg, 2.75 μmol, 1.0 eq) and BCY11014 (5.98 mg, 2.75 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 13.7 μl, 2.0 eq), VcNa (1.1 mg, 5.55 μmol, 2.0 eq), and THPTA (1.2 mg, 2.76 μmol, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 16 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 5810.66, observed m / z: 1163.0 ([M / 5+H]) +It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY10917 (6.4 mg, 1.07 μmol, 39.03% yield, 97.49% purity) was obtained as a white solid.

[0660] BCY11020

[0661]

[0662] Manufacturing process of BCY8831-PEG5-N3

[0663]

[0664] BCY8831 (25.0 mg, 8.31 μmol, 1.0 eq) and Compound 1 (3.9 mg, 9.02 μmol, 1.09 eq) were dissolved in DMF (1 mL). DIPEA (1.6 mg, 12.46 μmol, 2.2 μl, 1.5 eq) was added to the mixture, and the mixture was then stirred at 35°C for 2 hours. LC-MS confirmed that BCY8831 was completely eliminated and the target m / z (calculated MW: 3326.79, observed m / z: 1109.66 [M / 3+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (7.3 mg, 2.09 μmol, 25.20% yield, 95.41% purity) was obtained as a white solid.

[0665] BCY11020 manufacturing process

[0666]

[0667] Compound 2 (7.3 mg, 2.19 μmol, 1.0 eq) and BCY11014 (4.8 mg, 2.19 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 5.5 μl, 1.0 eq), VcNa (1.0 mg, 5.05 μmol, 2.3 eq), and THPTA (1.0 mg, 2.30 μmol, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 12 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 5502.29, observed m / z: 1101.74 ([M / 5+H]) + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY11020 (3.3 mg, 0.577 μmol, 26.30% yield, 96.24% purity) was obtained as a white solid.

[0668] BCY11373

[0669]

[0670] Preparation process of Compound 2

[0671]

[0672] EDCI (8.5 mg, 54.8 μmol, 1.1 eq) and HOSu (5.7 mg, 49.5 μmol, 1.0 eq) were added to a solution of Compound 1 (5.0 mg, 49.5 μmol, 1.0 eq) in DMF (1 mL). The mixture was stirred at 25-30°C for 30 minutes. TLC indicated that Compound 1 had completely disappeared and a new spot had formed. Subsequently, BCY8116 (30.0 mg, 13.81 μmol, 0.28 eq) and DIEA (2.4 μl, 13.81 μmol, 0.28 eq) were added to 0.3 mL of the mixture, and the mixture was stirred at 25-30°C for 2 hours. LC-MS showed that BCY8116 was completely extinguished and the target m / z (calculated MW: 2255.53, observed m / z: 1128.34 ([M / 2+H]) + It was shown that a single main peak having ) was detected. Subsequently, the reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue, which was then purified by pre-HPLC (under TFA conditions). Compound 2 (21 mg, 8.9 μmol, 64.43% yield, 95.56% purity) was obtained as a white solid.

[0673] Manufacturing process of BCY11373

[0674]

[0675] A mixture of Compound 2 (5 mg, 2.22 μmol, 1.0 eq), BCY8928 (4.79 mg, 2.22 μmol, 1.0 eq), and THPTA (1.0 mg, 2.30 μmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 5.6 μL, 1.0 eq) and VcNa (0.4 M, 5.6 μL, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 4415.07, observed m / z : 1471.5([M / 3+H] + and 1103.8([M / 4+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by pre-HPLC (TFA conditions), and BCY11373 (4.9 mg, 1.03 μmol, 46.26% yield, 92.4% purity) was obtained as a white solid.

[0676] BCY11374

[0677]

[0678] BCY11374 manufacturing process

[0679]

[0680] A mixture of compound 2 (which can be prepared as described in the preparation process of BCY11373; 5 mg, 2.22 μmol, 1.0 eq), BCY8928 (4.9 mg, 2.22 μmol, 1.0 eq), and THPTA (1.0 mg, 2.30 μmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 5.6 μl, 1.0 eq) and VcNa (0.4 M, 5.6 μl, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS indicated that Compound 2 was completely extinguished and the target m / z (calculated MW: 4473.11, observed m / z : 1491.5([M / 3+H] + and 1118.5([M / 4+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was directly purified by pre-HPLC (TFA conditions), and BCY11374 (4.1 mg, 1.27 μmol, 38.04% yield, 92.0% purity) was obtained as a white solid.

[0681] BCY11375

[0682]

[0683] BCY11375 manufacturing process

[0684]

[0685] A mixture of compound 2 (which can be prepared as described in the preparation process of BCY11373; 5 mg, 2.22 μmol, 1.0 eq), BCY11014 (4.8 mg, 2.22 μmol, 1.0 eq), and THPTA (0.5 mg, 2.30 μmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 5.6 μl, 1.0 eq) and VcNa (0.4 M, 5.6 μl, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS obtained some of the desired m / z (calculated MW: 4431.03, observed m / z : 1107.59([M / 4+H] + and 1477.90([M / 3+H] + ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was directly purified by pre-HPLC (TFA conditions), and BCY11375 (6 mg, 1.31 μmol, 59.13% yield, 96.8% purity) was obtained as a white solid.

[0686] BCY11616

[0687]

[0688] Preparation process of Compound 3

[0689]

[0690] A mixture of compound BCY8116 (30.0 mg, 13.81 μmol, 1.0 eq), compound 2 (6.0 mg, 13.88 μmol, 1.0 eq), and DIEA (2.4 μl, 13.82 μmol, 1.0 eq) was dissolved in DMF. LC-MS of the reaction mixture indicated that compound 1 was completely extinguished and the target m / z (calculated MW: 2389.82, observed m / z: 1245.4 [M / 2+H + The mixture was stirred at 40°C for 1 hour until a single main peak having ]) was detected. The reaction mixture was then concentrated under reduced pressure to remove the solvent and produce a residue, which was then purified by pre-HPLC (under TFA conditions). Compound 3 (27 mg, 10.29 μmol, 74.52% yield, 94.9% purity) was obtained as a white solid.

[0691] BCY11616 manufacturing process

[0692]

[0693] A mixture of Compound 3 (5 mg, 2.01 μmol, 1.0 eq), BCY7744 (5.2 mg, 2.21 μmol, 1.0 eq), and THPTA (1.0 mg, 2.30 μmol, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 5.0 μl, 1.0 eq) and VcNa (0.4 M, 5.0 μl, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS showed that compound 3 was completely extinguished and the target m / z (calculated MW: 4827.46, observed m / z : 1207.12([M / 4+H] +It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was directly purified by pre-HPLC (TFA conditions), and BCY11616 (4.7 mg, 1.0 μmol, 48.48% yield, 94.7% purity) was obtained as a white solid.

[0694] BCY11617

[0695]

[0696] BCY11617 manufacturing process

[0697]

[0698] A mixture of compound 3 (which can be prepared as described in the preparation process of BCY11616; 5 mg, 2.01 μmol, 1.0 eq), BCY11506 (5.2 mg, 2.21 μmol, 1.0 eq), and THPTA (1.0 mg, 2.30 μmol, 1.1 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 5.0 μl, 1.0 eq) and VcNa (0.4 M, 5.0 μl, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z (calculated MW: 4828.45, observed m / z : 1206.97([M / 4+H] + and 965.91([M / 5+H] +It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by pre-HPLC (TFA conditions), and BCY11617 (3.2 mg, 0.63 μmol, 31.37% yield, 95.05% purity) was obtained as a white solid.

[0699] BCY11857

[0700]

[0701] Manufacturing process of BCY11414-PEG5-N3

[0702]

[0703] BCY11414 (60.0 mg, 29.06 μmol, 1.0 eq) and Compound 1 (13.0 mg, 30.06 μmol, 1.03 eq) were dissolved in 2 mL of MeCN / H2O (1:1). The pH was adjusted to 8 with NaHCO3 (0.4 M), and the mixture was then stirred at 25–30 °C for 2 hours. LC-MS was performed on the target m / z (calculated MW: 2381.72, observed m / z: 1191.07 [M / 2+H]). + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (38.0 mg, 15.9 μmol, 54.71% yield, 97.35% purity) was obtained as a white solid.

[0704] Manufacturing process of BCY11857

[0705]

[0706] Compound 2 (10.0 mg, 4.20 μmol, 1.0 eq) and BCY7744 (11.5 mg, 4.92 μmol, 1.2 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 11.0 μl, 1.0 eq), VcNa (2.0 mg, 10 μmol, 2.4 eq), and THPTA (2.0 mg, 4.6 μmol, 1.1 eq). Finally, 0.2 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 16 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 4719.37, observed m / z: 1180.24 ([M / 4+H]) + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY11857 ​​(10.3 mg, 2.18 μmol, 51.90% yield, 96.02% purity) was obtained as a white solid.

[0707] BCY11858

[0708]

[0709] Manufacturing process of BCY11414-PEG5-N3

[0710]

[0711] BCY11414 (60.0 mg, 29.06 μmol, 1.0 eq) and Compound 1 (13.0 mg, 30.06 μmol, 1.03 eq) were dissolved in 2 mL of MeCN / H2O (1:1). The pH was adjusted to 8 with NaHCO3 (0.4 M), and the mixture was then stirred at 25–30 °C for 2 hours. LC-MS was performed on the target m / z (calculated MW: 2381.72, observed m / z: 1191.07 [M / 2+H]). +It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (38.0 mg, 15.9 μmol, 54.71% yield, 97.35% purity) was obtained as a white solid.

[0712] BCY11858 manufacturing process

[0713]

[0714] Compound 2 (20.0 mg, 8.40 μmol, 1.0 eq) and BCY8928 (22.0 mg, 9.92 μmol, 1.1 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 21.0 μl, 1.0 eq), VcNa (4.0 mg, 20.19 μmol, 2.4 eq), and THPTA (4.0 mg, 9.20 μmol, 1.1 eq). Finally, 0.4 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 16 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 4599.30, observed m / z: 920.38 ([M / 5+H]) + ), 1150.79([M / 4+H] + ), 1533.35([M / 3+H] + It was shown that a single main peak having ) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY11858 (16.9 mg, 3.67 μmol, 43.43% yield, 99.25% purity) was obtained as a white solid.

[0715] BCY11859

[0716]

[0717] Manufacturing process of BCY11415-PEG5-N3

[0718]

[0719] BCY11415 (30.0 mg, 13.81 μmol, 1.0 eq) and Compound 1 (6.0 mg, 30.06 μmol, 1.0 eq) were dissolved in 2 mL of MeCN / H2O (1:1). The pH was adjusted to 8 with NaHCO3 (0.4 M), and the mixture was then stirred at 25–30 °C for 2 hours. LC-MS yielded the target m / z (calculated MW: 2489.82, observed m / z: 1245.18 [M / 2+H]). + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (24.0 mg, 9.63 μmol, 69.7% yield, 99.28% purity) was obtained as a white solid.

[0720] BCY11859 manufacturing process

[0721]

[0722] Compound 2 (20.0 mg, 8.03 μmol, 1.0 eq) and BCY8928 (21.0 mg, 9.47 μmol, 1.1 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 21.0 μl, 1.0 eq), VcNa (4.0 mg, 2.5 eq), and THPTA (4.0 mg, 1.1 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 16 hours under an N2 atmosphere. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 4707.40, observed m / z: 941.7 ([M / 5+H]) + ), 1176.9([M / 4+H] + ), 1569.6([M / 3+H] +It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and BCY11859 (19.2 mg, 4.01 μmol, 49.87% yield, 98.22% purity) was obtained as a white solid.

[0723] Example 4: Synthesis of PD-L1 / CD137-binding heterotandem acyclic peptide

[0724] BCY8939

[0725]

[0726] General manufacturing process of BCY8939

[0727]

[0728] EDCI (89.3 mg, 466 μmol) was added to a solution of N3-PEG12-COOH (250 mg, 388 μmol) and HOSu (67.0 mg, 583 μmol) in DMA (4.5 mL) and DC (1.5 mL) while stirring at 20°C for 16 hours. LCMS showed that the target intermediate was fully formed. BCY7732 (854.97 mg, 388.37 μmol, 1 eq) and DIEA (186 mg, 1.44 mmol, 250 μl) were added to the mixture while stirring for an additional 5 hours at 20°C. LC-MS showed that BCY7732 was completely extinguished and a single main peak with the target mass was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) to yield compound BCY7859 as a white solid (621 mg, 200.58 μmol, 51.65% yield, 95% purity, TFA). Calculated MW: 2817.16, observed m / z: 942.7 [M / 3+H] +

[0729] General manufacturing process of BCY8939

[0730]

[0731] (2R)-2-[(1S)-1,2-dihydroxyethyl]-3,4-dihydroxyl-2H-furan-5-one (1M, 100 µL) and CuSO4 (1M, 30.0 µL) were added to a solution of BCY7859 (31.1 mg, 11.0 μmol) and BCY8938 (30.0 mg, 10.0 μmol) in DMF (2 mL) while stirring under a nitrogen atmosphere at 20°C for 2 hours. LCMS showed that BCY7859 was completely eliminated and a single main peak with the desired mass was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) to yield compound BCY8939 (16.1 mg, 2.72 μmol, 27.1% yield, 98.3% purity) as a white solid. Calculated MW: 5823.49, Observed m / z: 1165.4[M / 5+H] + , 971.0[M / 6+H] + , 832.9[M / 7+H] +

[0732] BCY10580

[0733]

[0734] Manufacturing process of BCY9172-PEG12-N3

[0735]

[0736] DIEA (9.25 mg, 71.58 μmol, 12.47 μl, 1.5 eq) was added to BCY9172 (100.0 mg, 47.72 μmol, 1 eq) and Compound 1 (40.0 mg, 54.00 μmol, 1.13 eq) in DMSO (2 mL). The mixture was stirred at 30°C for 12 hours. LC-MS confirmed that BCY9172 was completely eliminated and the target m / z (MW: 2721.12, observed m / z : 1361.07([M / 2+H] +It was shown that a single main peak having )) was detected. The reaction mixture was concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by pre-HPLC (neutral conditions). Compound 2 (48 mg, 17.44 μmol, 45.68% yield, 98.87% purity) was provided as a white solid.

[0737] Manufacturing process of BCY10580

[0738]

[0739] Compound 2 (20 mg, 7.35 μmol, 1.0 eq) and BCY10043 (23.1 mg, 7.35 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 18.4 µL, 1.0 eq), VcNa (0.4 M, 36.8 µL, 2.0 eq), and THPTA (0.4 M, 18.4 µL, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 4 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and at the target m / z (MW: 5855.74, observed m / z: 976.40 [M / 6+H]). + ) and 1171.67([M / 5+H] + It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (TFA conditions). BCY10580 (29 mg, 4.85 μmol, 65.95% yield, 97.879% purity) was obtained as a white solid.

[0740] BCY10581

[0741]

[0742] Manufacturing process of BCY9172-PEG12-N3

[0743]

[0744] DIEA (9.25 mg, 71.58 μmol, 12.47 μl, 1.5 eq) was added to BCY9172 (100.0 mg, 47.72 μmol, 1 eq) and Compound 1 (40.00 mg, 54.00 μmol, 1.13 eq) in DMSO (2 mL). The mixture was stirred at 30°C for 12 hours. LC-MS confirmed that BCY9172 was completely eliminated and the target m / z (MW: 2721.12, observed m / z : 1361.07([M / 2+H + It was shown that a single main peak having ]) was detected. The reaction mixture was filtered and concentrated under reduced pressure to remove the solvent and produce a residue. The residue was then purified by pre-HPLC (neutral conditions). Compound 2 (48 mg, 17.44 μmol, 45.68% yield, 98.87% purity) was provided as a white solid.

[0745] BCY10581 manufacturing process

[0746]

[0747] Compound 2 (12 mg, 4.41 μmol, 1 eq) and BCY10044 (14.08 mg, 4.41 μmol, 1 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 11.02 μL, 1 eq), VcNa (0.4 M, 22.05 μL, 2 eq), and THPTA (0.4 M, 10.04 μL, 1 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred at 30°C for 4 hours under an N2 atmosphere. LC-MS showed that Compound 3 was completely extinguished and the target m / z (MW: 5912.84, observed m / z: 985.90 ([M / 6+H]) + ) and 1183.28([M / 5+H] +It was shown that a single main peak having )) was detected. The residue was purified by pre-HPLC (TFA conditions). BCY10581 (9.3 mg, 1.47 μmol, 33.36% yield, 93.541% purity) was obtained as a white solid.

[0748] BCY10582

[0749]

[0750] Preparation process of Compound 2

[0751]

[0752] DIEA (9.2 mg, 71.6 μmol, 12.5 μl, 1.5 eq) was added to a solution of BCY9172 (100.0 mg, 47.7 μmol, 1.0 eq) and Compound 1 (40.0 mg, 54.0 μmol, 1.13 eq) in DMSO (2 mL). The mixture was stirred at 30°C for 12 hours. LC-MS confirmed that BCY9172 was completely eliminated and the target m / z (MW: 2721.12, observed m / z : 1361.07([M / 2+H] + It was shown that a single main peak having )) was detected. The reaction mixture was filtered and concentrated under reduced pressure to remove the solvent and provide the residue. The residue was purified by pre-HPLC (neutral conditions). Compound 2 (37 mg, 13.60 μmol, 28.49% yield) was obtained as a white solid.

[0753] BCY10582 manufacturing process

[0754]

[0755] A mixture of compound 2 (16.0 mg, 5.9 μmol, 1.0 eq), BCY10045 (14.0 mg, 6.0 μmol, 1.01 eq), and THPTA (0.4 M, 14.7 μl, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 2 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 14.7 μl, 1.0 eq) and VcNa (0.4 M, 29.4 μl, 2.0 eq) were added under N2. The pH of the solution was adjusted to 8 by adding 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 25–30°C for 12 hours under an N2 atmosphere. LC-MS confirmed that Compound 2 was completely extinguished and the target m / z [Calculated MW: 5073.89, Observed m / z: 1015.24 ([M / 5+H]] + ) and 1268.97([M / 4+H + It was shown that a single main peak having ])) was detected. The reaction mixture was directly purified by pre-HPLC (TFA conditions). BCY10582 (10 mg, 1.92 μmol, 32.58% yield, 97.21% purity) was obtained as a white solid.

[0756] BCY11017

[0757]

[0758] BCY11017 manufacturing process

[0759]

[0760] Compound 2 (which can be prepared as described in the preparation process of BCY10567; 7.0 mg, 2.59 μmol, 1.0 eq) and BCY10861 (7.03 mg, 2.59 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 13.0 μl, 2.0 eq), VcNa (1.0 mg, 5.03 μmol, 2.0 eq), and THPTA (1.1 mg, 2.53 μmol, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred under an N2 atmosphere at 35°C for 16 hours. LC-MS showed that Compound 2 was completely extinguished and the target m / z (calculated MW: 5421.30, observed m / z : 1084.7([M / 5+H] + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions), and BCY11017 (6.6 mg, 1.17 μmol, 45.24% yield, 96.16% purity) was obtained as a white solid.

[0761] BCY11018

[0762]

[0763] BCY11018 manufacturing process

[0764]

[0765] Compound 2 (which can be prepared as described in the preparation process of BCY10570; 6.0 mg, 2.17 μmol, 1.0 eq) and BCY10861 (5.9 mg, 2.17 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 11.0 μl, 2.0 eq), VcNa (1.0 mg, 2.3 eq), and THPTA (1.1 mg, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred under an N2 atmosphere at 35°C for 16 hours. LC-MS showed that Compound 2 was completely extinguished and the target m / z (calculated MW: 5479.34, observed m / z : 1096.40([M / 5+H] + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions), and BCY11018 (2.3 mg, 0.40 μmol, 18.31% yield, 94.73% purity) was obtained as a white solid.

[0766] BCY11019

[0767]

[0768] BCY11019 manufacturing process

[0769]

[0770] Compound 2 (which can be prepared as described in the preparation process of BCY10581; 8.0 mg, 2.59 μmol, 1.0 eq) and BCY10861 (8.0 mg, 2.95 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 14.7 μl, 2.0 eq), VcNa (1.2 mg, 6.05 μmol, 2.0 eq), and THPTA (1.3 mg, 2.99 μmol, 1.0 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred under an N2 atmosphere at 35°C for 16 hours. LC-MS showed that Compound 2 was completely extinguished and the target m / z (calculated MW: 5437.26, observed m / z : 1088.09([M / 5+H] + ) and 1360.19([M / 4+H] + It was shown that a single main peak having ) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions), and BCY11019 (7.6 mg, 1.36 μmol, 46.09% yield, 96.95% purity) was obtained as a white solid.

[0771] BCY11376

[0772]

[0773] Preparation process of Compound 2

[0774]

[0775] EDCI (8.5 mg, 54.8 μmol, 1.1 eq) and HOSu (5.7 mg, 49.5 μmol, 1.0 eq) were added to a solution of Compound 1 (5.0 mg, 49.5 μmol, 1.0 eq) in DMF (1 mL). The mixture was stirred at 25-30°C for 30 minutes. TLC indicated that Compound 1 had completely disappeared and a new spot had formed. Subsequently, BCY8919 (20.0 mg, 9.62 μmol) and DIEA (1.7 μl, 9.62 μmol) were added to 0.2 mL of the above mixture. The mixture was stirred at 25-30°C for 2 hours. LC-MS showed that BCY8919 was completely extinguished and the target m / z (calculated MW: 2162.51, observed m / z: 1081.8 ([M / 2+H]) + It was shown that a single main peak having ) was detected. Subsequently, the reaction mixture was concentrated under reduced pressure to remove the solvent and generate a residue, which was then purified by pre-HPLC (under TFA conditions). Compound 2 (12 mg, 5.55 μmol, 56.28% yield, 97.54% purity) was obtained as a white solid.

[0776] BCY11376 manufacturing process

[0777]

[0778] A mixture of Compound 2 (3 mg, 1.39 μmol, 1.0 eq), BCY10861 (3.8 mg, 1.40 μmol, 1.0 eq), and THPTA (1.2 mg, 2.76 μmol, 2.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 3.5 μl, 1.0 eq) and VcNa (0.4 M, 3.5 μl, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS showed that BCY10861 had completely faded and the target m / z (calculated MW: 4878.64, observed m / z : 1220.8([M / 4+H] + It was shown that a single main peak having )] was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was directly purified by pre-HPLC (TFA conditions), and BCY11376 (1.9 mg, 1.0 μmol, 27.01% yield, 96.2% purity) was obtained as a white solid.

[0779] BCY11377

[0780]

[0781] Preparation process of Compound 2

[0782]

[0783] EDCI (8.5 mg, 54.8 μmol, 1.1 eq) and HOSu (5.7 mg, 49.5 μmol, 1.0 eq) were added to a solution of Compound 1 (5.0 mg, 49.5 μmol, 1.0 eq) in DMF (1 mL). The mixture was stirred at 25-30°C for 30 minutes. TLC indicated that Compound 1 had completely disappeared and a new spot had formed. Subsequently, BCY8920 (20.0 mg, 9.36 μmol) and DIEA (1.2 mg, 9.36 μmol) were added to 0.2 mL of the above mixture. The mixture was stirred at 25-30°C for 2 hours. LC-MS showed that BCY8920 was completely extinguished and the target m / z (calculated MW: 2220.54, observed m / z: 1110.90 ([M / 2+H]) + It was shown that a single main peak having ) was detected. Subsequently, the reaction mixture was concentrated under reduced pressure to remove the solvent and generate a residue, which was then purified by pre-HPLC (under TFA conditions). Compound 2 (12 mg, 5.15 μmol, 56.28% yield, 95.3% purity) was obtained as a white solid.

[0784] Manufacturing process of BCY11377

[0785]

[0786] A mixture of Compound 2 (3 mg, 1.35 μmol, 1.0 eq), BCY10861 (3.8 mg, 1.35 μmol, 1.0 eq), and THPTA (0.6 mg, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 3.4 μl, 1 eq) and VcNa (0.4 M, 3.4 μl, 1 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS is the target m / z (calculated MW: 4936.68, observed m / z : 1234.9([M / 4+H] + It was shown that a single main peak having )] was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was directly purified by pre-HPLC (TFA conditions), and BCY11377 (3.5 mg, 0.66 μmol, 48.86% yield, 93.1% purity) was obtained as a white solid.

[0787] BCY11378

[0788]

[0789] Preparation process of Compound 2

[0790]

[0791] EDCI (8.5 mg, 54.8 μmol, 1.1 eq) and HOSu (5.7 mg, 49.5 μmol, 1.0 eq) were added to a solution of Compound 1 (5.0 mg, 49.5 μmol, 1.0 eq) in DMF (1 mL). The mixture was stirred at 25-30°C for 30 minutes. TLC indicated that Compound 1 had completely disappeared and a new spot had formed. Subsequently, 0.2 mL of the above mixture was added to BCY9172 (20.0 mg, 9.54 μmol) and DIEA (1.7 μl, 9.62 μmol). The mixture was stirred at 25-30°C for 2 hours. LC-MS showed that Compound 1 was completely extinguished and the target m / z (calculated MW: 2176.49, observed m / z: 1090.0 ([M / 2+H]) + It was shown that a single main peak having ) was detected. Subsequently, the reaction mixture was concentrated under reduced pressure to remove the solvent and generate a residue, which was then purified by pre-HPLC (under TFA conditions). Compound 2 (20.2 mg, 7.48 μmol, 78.34% yield, 80.57% purity) was obtained as a white solid.

[0792] Manufacturing process of BCY11378

[0793]

[0794] A mixture of Compound 2 (5 mg, 2.30 μmol, 1.0 eq), BCY10861 (6.24 mg, 2.30 μmol, 1.0 eq), and THPTA (1.0 mg, 1.0 eq) was dissolved in t-BuOH / H2O (1:1, 1 mL, pre-degassed and purged three times with N2), and then CuSO4 (0.4 M, 5.8 μL, 1.0 eq) and VcNa (0.4 M, 5.8 μL, 1.0 eq) were added under N2. The pH of the solution was adjusted to 8 by the titration of 0.2 M NH4HCO3 (in 1:1 t-BuOH / H2O), and the solution turned bright yellow. The reaction mixture was stirred at 40°C for 2 hours under an N2 atmosphere. LC-MS showed that compound 3 was completely extinguished and the target m / z (calculated MW: 4894.61, observed m / z : 1224.3([M / 4+H] + It was shown that a single main peak having ) was detected. The reaction mixture was filtered and concentrated under reduced pressure to provide the residue. The crude product was purified by pre-HPLC (TFA conditions), and BCY11378 (1.2 mg, 0.34 μmol, 10.07% yield, 94.3% purity) was obtained as a white solid.

[0795] BCY11379

[0796]

[0797] Manufacturing process of BCY8919-PEG5-N3

[0798]

[0799] BCY8919 (30.0 mg, 14.43 μmol, 1.0 eq) and Compound 1 (6.3 mg, 14.57 μmol, 1.01 eq) were dissolved in a mixture of MeCN (1 mL) and H2O (1 mL). 1 M NaHCO3 was added to the solution to adjust the pH to 8, and the mixture was then stirred at 35°C for 2 hours. LC-MS confirmed that BCY8919 was completely eliminated and the target m / z (calculated MW: 2396.79, observed m / z: 1198.74 [M / 2+H]) was achieved. + ) and 799.50([M / 4+H] + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (20 mg, 8.07 μmol, 55.92% yield, 96.68% purity) was obtained as a white solid.

[0800] BCY11379 manufacturing process

[0801]

[0802] Compound 2 (3.0 mg, 1.25 μmol, 1.0 eq) and BCY10861 (3.4 mg, 1.25 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 7 μL, 2.24 eq), VcNa (1 mg, 5.04 μmol, 4.03 eq), and THPTA (1 mg, 2.30 μmol, 1.84 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred under an N2 atmosphere at 25-30°C for 16 hours. LC-MS showed that compound 2 was completely extinguished and the target m / z (calculated MW: 5112.93 observed m / z : 1022.96([M / 5+H] + ) and 1278.74([M / 4+H] +It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions), and BCY11379 (3.4 mg, 0.615 μmol, 52.00% yield, 97.88% purity) was obtained as a white solid.

[0803] BCY11380

[0804]

[0805] Manufacturing process of BCY8920-PEG5-N3

[0806]

[0807] BCY8920 (30.0 mg, 14.04 μmol, 1.0 eq) and Compound 1 (6.1 mg, 14.11 μmol, 1.01 eq) were dissolved in a mixture of MeCN (1 mL) and H2O (1 mL). 1 M NaHCO3 was added to the solution to adjust the pH to 8, and the mixture was then stirred at 35°C for 2 hours. LC-MS confirmed that BCY8920 was completely eliminated and the target m / z (calculated MW: 2454.83, observed m / z: 1227.63 [M / 2+H]) was achieved. + ) and 818.66([M / 3+H] + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (20 mg, 8.03 μmol, 57.21% yield, 98.56% purity) was obtained as a white solid.

[0808] Manufacturing process of BCY11380

[0809]

[0810] Compound 2 (3.5 mg, 1.43 μmol, 1.0 eq) and BCY10861 (3.9 mg, 1.44 μmol, 1.0 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 8 μL, 2.24 eq), VcNa (1 mg, 5.04 μmol, 3.52 eq), and THPTA (1 mg, 2.30 μmol, 1.61 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred under an N2 atmosphere at 25-30°C for 16 hours. LC-MS showed that Compound 2 was completely extinguished and the target m / z (calculated MW: 5170.97, observed m / z : 1034.28([M / 5+H] + ) and 1293.10([M / 4+H] + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions), and BCY11380 (1.6 mg, 0.296 μmol, 20.77% yield, 96.77% purity) was obtained as a white solid.

[0811] BCY11381

[0812]

[0813] Manufacturing process of BCY8920-PEG5-N3

[0814]

[0815] BCY9172 (30.0 mg, 14.32 μmol, 1.0 eq) and Compound 1 (6.2 mg, 14.34 μmol, 1.0 eq) were dissolved in a mixture of MeCN (1 mL) and H2O (1 mL). 1 M NaHCO3 was added to the solution to adjust the pH to 8, and the mixture was then stirred at 35°C for 2 hours. LC-MS confirmed that BCY9172 was completely eliminated and the target m / z (calculated MW: 2412.75, observed m / z: 1206.72 [M / 2+H]) was achieved. + It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions) and compound 2 (15 mg, 6.14 μmol, 42.87% yield, 98.75% purity) was obtained as a white solid.

[0816] BCY11381 manufacturing process

[0817]

[0818] Compound 2 (3.0 mg, 1.24 μmol, 1.0 eq) and BCY10861 (3.4 mg, 1.25 μmol, 1.01 eq) were first dissolved in 2 mL of t-BuOH / H2O (1:1), followed by the addition of CuSO4 (0.4 M, 7 μL, 2.25 eq), VcNa (1 mg, 5.04 μmol, 4.06 eq), and THPTA (1 mg, 2.30 μmol, 1.85 eq). Finally, 1 M NH4HCO3 was added to adjust the pH to 8. At this point, all solvents were degassed and purged with N2 three times. The reaction mixture was stirred under an N2 atmosphere at 25-30°C for 16 hours. LC-MS is the target m / z (calculated MW: 5128.89, observed m / z : 1026.05([M / 5+H] + ) and 1282.50([M / 4+H] +It was shown that a single main peak having )) was detected. The reaction mixture was purified by pre-HPLC (TFA conditions), and BCY11381 (1.6 mg, 0.295 μmol, 23.73% yield, 94.59% purity) was obtained as a white solid.

[0819] Example 5: Production of CD137 Monoclonal Antibody Agent

[0820] The sequence of the CD137 monoclonal antibody agent used for comparison with CD137 multimers in the experiments provided herein is disclosed in U.S. Patent No. 7,288,638. IgG4 isotype antibodies were expressed using the ExpiCHO expression system (Thermo Fisher Scientific) following transient transfection of DNA expression constructs. The antibodies were purified by protein A affinity chromatography and formulated in phosphate-buffered saline (PBS) at pH 7.2. Purity analysis using HPLC-SEC (Column GF-250, Agilent) indicated that the monomer ratio of the CD137 monoclonal antibody was approximately 95%. Binding activity analysis indicated that the CD137 monoclonal antibody at a concentration greater than 1 µg / ml could bind to CHO cells expressing CD137. Toxin Sensor TM Endotoxin analysis using the chromogenic LAL endotoxin assay kit (Genscript) indicated that the CD137 monoclonal antibody preparation contained <7 EU / mg of endotoxin.

[0821] biological data

[0822] 1. CD137 ViaCore Experiment Description

[0823] Biacore experiments were performed on k heterotandem peptides binding to the human CD137 protein. a (M -1 s -1 ), k d (s -1 ), K DThe (nM) value was measured. Recombinant human CD137 (R&D systems) EZ-Link TM Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) was resuspended in PBS and biotinated according to the manufacturer's protocol. The protein was desalted into PBS using a rotary column to remove unbound biotin.

[0824] For protein binding analysis, Viacore T200 or Viacore 3000 devices equipped with a XanTec CMD500D chip were used. Streptavidin was immobilized on the chip using standard amine-binding chemistry with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer at 25°C. Briefly, the carboxymethyl dextran surface was activated by a 7-minute injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 µl / min. For the capture of streptavidin, the protein was diluted to 0.2 mg / mL in 10 mM sodium acetate (pH 4.5), and 120 µL was injected onto the activated chip surface for capture. Residual activators were blocked by injecting 1 M ethanolamine (pH 8.5) for 7 minutes, and biotinylated CD137 was captured at levels of 270–1500 RU. The buffer was exchanged with PBS / 0.05% Tween 20, and serial dilutions of the peptide were prepared in the said buffer to a final DMSO concentration of 0.5%. The normal peptide concentration was 500 nM with six additional 2-fold or 3-fold dilutions. SPR analysis was performed at 25°C with a flow rate of 90 µL / min, with 60 seconds of binding and 900 seconds of dissociation. A regeneration step (10 µL of 10 mM glycine pH 2) was used after each cycle. The data were corrected for volume effects excluding DMSO as needed. All data were double-referenced against blank injection and reference surfaces using standard processing procedures, and data processing and kinetic fitting were performed using Scrubber software, version 2.0c (BioLogic Software). Where the data were suitable, a simple 1:1 coupled model allowing for mass transfer effects was fitted.

[0825] Several heterotandem peptides were tested in the above analysis, and the results are shown in Table 1 below.

[0826] CD137 Viacore analysis data for heterotandem peptides Complex ID SPR (K D )(nM) BCY9173 7.98 BCY7985 143 BCY8942 853 BCY8943 156 BCY9647 206 BCY9648 202 BCY9655 199 BCY9656 159 BCY9657 256 BCY9658 152 BCY9659 88.1 BCY9758 189 BCY8854 108 BCY9350 69.4 BCY9351 3640 BCY9399 73 BCY9400 53 BCY9408 105 BCY9409 97.7 BCY9410 65.8 BCY9411 71.1 BCY9759 44.3 BCY10000 6.19 BCY10571 12.03 BCY10572 5.00 BCY10573 3.39

[0827] 2. Description of the Nectin-4 Viacor Experiment

[0828] By performing Viacore experiments, k of heterotandem peptides binding to human nectin-4 protein (obtained from Charles River) a (M -1 s -1 ), k d (s -1 ), K D The (nM) value was measured.

[0829] Human nectin-4 (residue Gly32-Ser349; NCBI RefSeq: NP_112178.2) with gp67 signal sequence and C-terminal FLAG tag as a standard Bac-to-Bac TM It was cloned into pFastbac-1 and baculovirus prepared using the Life Technologies protocol. 1 x 10⁶ in Excell-420 medium (Sigma) 6 ml -1 Sf21 cells were infected with P1 virus stock at an MOI of 2 at 27°C, and the supernatant was harvested at 72 hours. The supernatant was batch bound with Anti-FLAG M2 affinity agarose resin (Sigma), washed in PBS, at 4°C for 1 hour; subsequently, the resin was transferred to a column and extensively washed with PBS. Proteins were eluted with 100 µg / ml FLAG peptide. The eluted protein was concentrated to 2 ml and loaded onto an S-200 Superdex column (GE Healthcare) in PBS at a rate of 1 ml / min. The 2 ml fraction was collected, and the fraction containing nectin-4 protein was concentrated to 16 mg / ml.

[0830] Protein EZ-Link TMThe protein was randomly biotinized in PBS using Sulfo-NHS-LC-LC-Biotin reagent (Thermo Fisher) according to the manufacturer's protocol. The protein was desalted into PBS using a rotary column to remove unbound biotin.

[0831] For protein binding analysis, a Viacore 3000 device equipped with a CM5 chip (GE Healthcare) was used. Streptavidin was immobilized on the chip using standard amine-binding chemistry with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer at 25°C. Briefly, the carboxymethyl dextran surface was activated by a 7-minute injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 µl / min. For the capture of streptavidin, the protein was diluted to 0.2 mg / mL in 10 mM sodium acetate (pH 4.5), and 120 µL of streptavidin was injected onto the activated chip surface for capture. Residual activators were blocked by injecting 1 M ethanolamine (pH 8.5) for 7 minutes, and biotinylated nectin-4 was captured at levels of 1,200–1,800 RU. The buffer was exchanged with PBS / 0.05% Tween 20, and serial dilutions of the peptide were prepared in the buffer to a final DMSO concentration of 0.5%. The normal peptide concentration was 100 nM with six additional 2-fold dilutions. SPR analysis was performed at 25°C with a flow rate of 50 µL / min, with 60-second binding and 400–1,200-second dissociation depending on the individual peptide. Data were corrected for volume effects excluding DMSO. All data were double-referenced against blank injection and reference surfaces using standard processing procedures, and data processing and kinetic fitting were performed using scrubber software, version 2.0c (BioLogic Software). Where data were fitted, a simple 1:1 coupled model allowing for mass transfer effects was used for fitting.

[0832] Several heterotandem peptides of the present invention were tested in the aforementioned nectin-4 binding assay, and the results are shown in Table 2 below.

[0833] Nectin-4 Viacore analysis data for heterotandem peptides Complex ID SPR K D (nM) BCY8854 2.76 BCY9350 > 200 nM BCY9351 2.47 BCY9399 1.67 BCY9400 1.8 BCY9408 1.57 BCY9409 1.66 BCY9410 1.49 BCY9411 1.48 BCY9759 2.14 BCY10000 2.26

[0834] 3. EphA2 Viacore Experiment Description

[0835] By performing Viacore experiments, k of heterotandem peptides binding to human EphA2 protein a (M -1 s -1 ), k d (s -1 ), K D The (nM) value was measured.

[0836] EZ-Link with 3x molar excess biotin per protein for 1 hour in 4 mM sodium acetate, 100 mM NaCl, pH 5.4 TMBiotinization was performed with sulfo-NHS-LC-biotin. The degree of labeling was measured using a fluorescent biotin quantification kit (Thermo) after dialyzing the reaction mixture into PBS. For peptide bond analysis, a Viacore T200 device equipped with a XanTec CMD500D chip was used. Streptavidin was immobilized on the chip using standard amine-bonding chemistry at 25°C with HBS-N (10 mM HEPES, 0.15 M NaCl, pH 7.4) as the running buffer. Briefly, the carboxymethyl dextran surface was activated by a 7-minute injection of a 1:1 ratio of 0.4 M 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) / 0.1 M N-hydroxysuccinimide (NHS) at a flow rate of 10 µl / min. For the capture of streptavidin, the protein was diluted to 0.2 mg / mL in 10 mM sodium acetate (pH 4.5), and 120 µL was injected onto the activated chip surface for capture. Residual activators were blocked by injecting 1 M ethanolamine (pH 8.5):HBS-N (1:1) for 7 minutes. The buffer was exchanged with PBS / 0.05% Tween 20, and biotinylated EphA2 was captured at levels of 500–1500 RU using a protein dilution of 0.2 μM in the buffer. Serial dilutions of the peptide were prepared in the buffer at a final DMSO concentration of 0.5%, where the normal peptide concentration was 50 or 100 nM with six additional 2-fold dilutions. SPR analysis was performed at 25°C with a flow rate of 90 µL / min, with 60 seconds of binding and 900–1200 seconds of dissociation. The data were corrected for volume effects excluding DMSO. All data were double-referenced against blank injection and reference surfaces using standard processing procedures, and data processing and kinetic fitting were performed using scrubber software, version 2.0c (BioLogic Software). Where the data were fitted, a simple 1:1 coupled model allowing for mass transfer effects was used.

[0837] Several heterotandem peptides of the present invention were tested in EphA2 binding analysis, and the results are shown in Table 3 below.

[0838] EphA2 Viacore analysis data for heterotandem peptides Complex ID SPR K D (nM) BCY9173 2.1 BCY7985 2 BCY8942 1.7 BCY8943 > 200 nM BCY9647 1.69 BCY9648 1.75 BCY9655 1.33 BCY9656 0.75 BCY9657 1.1 BCY9658 1.9 BCY9659 1.03 BCY9758 1.5

[0839] 4. Analysis of CD137 Reporter Co-culture with Tumor Cells

[0840] Prepare the culture medium designated as R1 medium by adding 1% FBS to RPMI-1640 (a component of Promega Kit CS196005). Prepare a series of dilutions of the test item in R1 in a sterile 96-well plate. Use 25 µl of the test item or R1 (as a background control) per well in the designated wells of the leukocyte culture plate. Tumor cells * Harvest and resuspend in R1 medium at a concentration of 400,000 cells / mL. Use 25 µl / well of tumor cells in a leukocyte culture plate. Thaw Zurcat cells (Promega Kit CS196005, 0.5 mL) in a water bath and then add them to 5 mL of preheated R1 medium. Use 25 µl / well of Zurcat cells in a leukocyte culture plate. Incubate the cells and test items at 37°C, 5% CO2 for 6 hours. At the end of 6 hours, add 75 µl / well of Bio-Glo TM (Promega) is added and incubated for 10 minutes before reading luminescence on a plate reader (Clariostar, BMG). Calculate the change factor for the cells (Zurcat cells + cell line used for co-culture) and plot the result against the EC50 (nM) and background (Max) on a graphpad prism.

[0841] The tumor cell types used in co-culture vary depending on the tumor target specific to the heterotandem, as shown in Table 4 below.

[0842] Cell lines used for each tumor target Tumor Target Cell lines used for co-culture EphA2 A549, SC-OV-3, PC3, LNCaP Nectin-4 HT1376, NCI-H292 PD-L1 RKO

[0843] Figure 3 provides data indicating that the EphA2-CD137 heterotandem BCY7985 showed strong induction of CD137 cell activity in the Promega CD137 luciferase reporter assay in the presence of EphA2-expressing HT1080 cells. In the absence of HT1080 cells, there is no CD137 induction by the heterotandem.

[0844] Figure 4 provides data indicating that the EphA2 / CD137 heterotandem induces strong CD137 activation in CD137 reporter assays and that the activation-induced duplicating factor depends on the tumor target expression levels (A549 and SC-OV-3:EphA2 high and LNCaP:EphA2 low) in the cell lines used for co-culture.

[0845] Figure 6 provides data indicating that the nectin-4 / CD137 heterotandem induces strong CD137 activation in CD137 reporter assays and that the activation-induced duplicating factor depends on the tumor target expression levels (HT1376:nectin-4 high and NCI-H292:nectin-4 medium) in the cell lines used for co-culture.

[0846] Figure 9 provides data indicating that the PD-L1 / CD137 heterotandem induces strong activation of CD137 in the CD137 reporter assay in the presence of PD-L1-expressing cell lines. A summary of the EC50 (nM) and induction folds induced by the heterotandem peptide in the CD137 reporter assay during co-culture with different cell lines is reported in Table 5 below:

[0847] Induced drainage induced by heterotandem peptides in CD137 reporter analysis Complex ID Tumor Target Cell lines used for co-culture EC50 (nM) Derivative multiplier for the background BCY9173 EphA2 SC-OV-3 0.94 21 BCY7985 EphA2 SC-OV-3 4.0 15 BCY8942 EphA2 PC3 - <2x induction at 100 nM BCY8943 EphA2 PC3 - <2x induction at 100 nM BCY9647 EphA2 SC-OV-3 7.2 24 BCY9648 EphA2 SC-OV-3 9.3 20 BCY9655 EphA2 SC-OV-3 4.1 6 BCY9656 EphA2 SC-OV-3 1.1 3 BCY9657 EphA2 SC-OV-3 9.0 26 BCY9658 EphA2 SC-OV-3 6.2 11 BCY9659 EphA2 SC-OV-3 9.9 7 BCY9758 EphA2 SC-OV-3 1.2 7 BCY10568 EphA2 PC3 0.25 32 BCY10570 EphA2 PC3 0.41 38 BCY10574 EphA2 PC3 1.0 32 BCY10575 EphA2 PC3 0.62 38 BCY10576 EphA2 PC3 0.51 38 BCY10577 EphA2 PC3 0.28 37 BCY8854 Nectin 4 H1376 1.2 30 BCY9350 Nectin 4 H1376 - <2x induction at 100 nM BCY9351 Nectin 4 H1376 - <2x induction at 100 nM BCY9399 Nectin 4 H1376 11 13 BCY9400 Nectin 4 H1376 2.9 13 BCY9401 Nectin 4 H1376 18 70 BCY9407 Nectin 4 H1376 3.4 29 BCY9408 Nectin 4 H1376 1.1 20 BCY9409 Nectin 4 H1376 1.2 24 BCY9410 Nectin 4 H1376 1.3 24 BCY9411 Nectin 4 H1376 14 41 BCY9759 Nectin 4 H1376 2.7 15 BCY10000 Nectin 4 H1376 0.58 61 BCY10567 Nectin 4 H1376 1.7 45 BCY10569 Nectin 4 H1376 1.2 52 BCY10571 Nectin 4 H1376 3.5 60 BCY10572 Nectin 4 H1376 0.44 55 BCY10573 Nectin 4 H1376 0.90 55 BCY10578 Nectin 4 H1376 0.42 58 BCY10917 Nectin 4 H1376 0.27 54 BCY11020 Nectin 4 H1376 0.26 47 BCY11373 Nectin 4 H1376 0.16 74 BCY11374 Nectin 4 H1376 0.091 72 BCY11375 Nectin 4 H1376 0.23 72 BCY8939 Mouse PD-L1 MC38 - <2x induction at 100 nM BCY10580 PD-L1 RKO 28 3 BCY10581 PD-L1 RKO 18 6 BCY10582 PD-L1 RKO 28 4 BCY11017 PD-L1 RKO 66 4 BCY11018 PD-L1 RKO 27 7 BCY11019 PD-L1 RKO 18 6 BCY11376 PD-L1 RKO 127 9 BCY11377 PD-L1 RKO 40 6 BCY11378 PD-L1 RKO 80 3 BCY11379 PD-L1 RKO 68 6 BCY11380 PD-L1 RKO 34 7 BCY11381 PD-L1 RKO 105 7

[0848] 5. Primary Human T Cell-A549 Co-culture (Tumor Cell Apoptosis)

[0849] PBMCs were isolated from three healthy donors and nucleite red-labeled tumor target cells (human lung carcinoma cells A549) were tested in limited ratios in the presence of two concentrations of anti-CD3 stimulation. ®, ATCC CLL-185 TM Tumor cells: PBMC co-cultures were cultured with reed-bicycle at three concentrations. All test conditions were also plated onto tumor cells in the absence of stimulated PBMCs to detect direct tumor cell cytotoxicity. Tumor death was evaluated by counting viable Nucleite Red-positive tumor cells over time. Additionally, apoptotic tumor cells were identified using the Caspase 3 / 7 dye. Cultures were analyzed using an IncuCyte S3 machine that allows real-time live cell fluorescence imaging. Co-cultures were imaged for 72 hours. Each condition was established by inducing three cycles.

[0850] Figure 5 provides data demonstrating that the EphA2 / CD137 heterotandem induces tumor cell death in a primary human T-cell and cancer cell co-culture assay. An anti-CD137 mAb agonist is used as a control.

[0851] 6. Analysis of Human PBMC-4T1 Co-culture (Cytokine Release)

[0852] Mouse mammary gland tumor cell lines 4T1-1 (4T1-Mo) and mouse nectin-4 overexpressing 4T1 (4T1-D02) were cultured in RPMI1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 IU / mL penicillin and 100 IU / streptomycin, 20 mM HEPES, 1X non-essential amino acids, and 2 mM L-glutamine (RPMI running medium). Frozen PBMCs from healthy human donors were thawed, washed once in room temperature PBS, and then resuspended in RPMI running medium. For tumor cell and PBMC co-culture, 10,000 PBMCs and 2,000 tumor cells (5:1) were mixed and plated into each well of a 384-well plate. To stimulate human PBMCs, 125 ng / mL of soluble anti-CD3 mAb (clone OKT3) was added to the cultures on Day 0. Test, control, or vehicle control compounds were added to each well, making the final volume per well 100 µl. Plates were incubated in a 37°C cell culture incubator with 5% CO2 for 3 days or less. The supernatant was collected at 48 hours after stimulation, and human IL-2 and IFNγ were detected using the HTRF assay. The raw data were analyzed using Excel or PRIZM software to generate standard curves and interpolate protein concentrations. The data represent a single study of three different donor PBMCs tested in experimental replicates.

[0853] The data provided in Figure 7 demonstrates that the nectin-4 / CD137 heterotandem induces robust IL-2 and IFN-γ cytokine secretion in a PBMC-4T1 co-culture assay. BCY9350 and BCY9351 are non-conjugated controls for nectin-4 and CD137, respectively.

[0854] A summary of EC50 (nM) and maximum IFN-γ cytokine secretion (pg / mL) induced by selected nectin-4 / CD137 heterotandem peptides in the human PBMC-4T1 co-culture (cytokine release) assay is reported in Table 6 below:

[0855] EC50 and maximum IFN-γ cytokine secretion induced by selected nectin-4 / CD137 heterotandem peptides in human PBMC-4T1 co-culture (cytokine release) assay Complex ID cell line EC50 (nM) max IFN-y (pg / ml) BCY8854 4T1-D02(Nectin 4+) 0.89 15962 BCY9350 4T1-D02(Nectin 4+) - No activity up to 1 μM BCY9351 4T1-D02(Nectin 4+) - No activity up to 1 μM BCY10000 4T1-D02(Nectin 4+) 0.21 19642 BCY10571 4T1-D02(Nectin 4+) 0.44 18349 BCY10572 4T1-D02(Nectin 4+) 0.25 17915

[0856] 7. In Vivo Culture Protocol

[0857] Primary patient-derived tumor cells from Discovery Life Sciences (DLS) are slowly thawed in 10 mL of pre-warmed wash medium freshly supplemented with benzonase. Cells are maintained in the culture for 2 days using a Greiner 3D Spheroid Kit (cat#655840). Briefly, tumor cells are counted with trypan blue using a hemocytometer. Cells are washed by centrifugation at 1500 rpm for 5 minutes, and the pellet is collected at a volume of 100 µL / 1 x 10⁶ 6Resuspend the cells in the N3D nanoshuttle. To make the above magnetic, spin the cells at 1500 rpm for 5 minutes and resuspend them; repeat the above process a total of 4 times. After the final spin, resuspend the cells in an appropriate amount of fresh spent DTC medium (DLS) to provide 50,000-100,000 cells per well at 100 µl / well. Use a Greiner cell-repulsion 96-well plate (cat#655976) for the above experiment. If cell clumps or debris are visible, apply the sample to a 70-100 µm filter before plating. Reserve at least 50,000 cells per sample for the Day 0 cytology panel, stain and fix the cells, and store at 4°C for later cytology analysis. A control / test compound dilution is prepared in a separate plate at 2x in lung DTC medium, and 100 µl / well of the 2X drug solution is added to the wells as indicated by the plate map. The analysis plate is then placed on a 96-well magnetic spheroid drive in a humidified chamber at 37°C and 5% CO2. At 24h, the magnetic spheroid drive is removed. At 48h, the medium is collected for cytokine analysis and cells are collected for the Day 2 cytology panel. Cytokines are quantified on a Luminex reader using a custom-made cytokine / chemokine panel from R&D Systems (IP-10, Granzyme B, IFNγ, IL-2, IL-6, TNFα, IL-8, MIP-1a, MIP-1b, MCP-1, IL-10, MIG). Knowledge panel: Day 0 = live / dead, CD45, EpCAM, Nectin4, CD3, CD4, CD8, CD137; Day 2 = live / dead, CD45, EpCAM, Nectin4, CD3, CD8, Ki67, and counting beads. Analyze knowledge data using Flowjo software.

[0858] Data demonstrating that the nectin-4 / CD137 heterotandem induces target-dependent cytokine release in in vitro cultures of primary patient-derived lung tumors are shown in Fig. 8. Treatment with BCY10572 showed multiple immune markers (normalized to the vehicle) and %CD8 in patient-derived samples + ki67 + Induced nectin-4-dependent changes in T cells.

[0859] 8. Pharmacokinetics of CD137 bispecificity in SD rats

[0860] Male SD rats were administered 2 mg / kg of each bicycle multimer formulated in 25 mM histidine-HCl and 10% sucrose pH 7. A series of hemorrhages (approx. 80 µl blood / time) were performed via the submandibular or saphenous vein at each time point. All blood samples were immediately transferred to a pre-cooled microcentrifuge tube containing 2 µl of K2-EDTA (0.5 M) as an anticoagulant and placed on wet ice. The blood samples were immediately treated against the plasma by centrifugation at approximately 4°C and 3000 g. A precipitating agent containing an internal standard was immediately added to the plasma, mixed well, and centrifuged at 12,000 rpm at 4°C for 10 minutes. The supernatant was transferred to a pre-labeled polypropylene microcentrifuge tube and subsequently flash-frozen on dry ice. Samples were stored at 70°C or below as needed until analysis. A 7.5 µL supernatant sample was directly injected for LC-MS / MS analysis using an Orbitrap Q Exactive in cation mode. Plasma concentration versus time data were analyzed by a non-compartmental approach using the Phoenix WinNonlin 6.3 software program. Graphs of CO, Cl, Vdss, T½, AUC(0-last), AUC(0-inf), MRT(0-last), MRT(0-inf), and plasma concentration versus time profiles were reported.

[0861] Figure 10 shows the plasma concentration versus time curves of BCY10572 and BCY10000 from a 2 mg / kg IV dose in SD rats (n=3):

[0862] Pharmacokinetic parameters of plasma concentration versus time curves of BCY10572 and BCY10000 compound T1 / 2(h) Clp (㎖ / min / ㎏) Vdss (L / kg) BCY10000 0.357 16.1 0.395 BCY10572 0.926 15.6 0.882

Claims

Claim 1 (b) a bicyclic peptide complex comprising a first peptide ligand that binds to a component present on an immune cell, which is conjugated via a linker to a second peptide ligand that binds to a component present on a cancer cell, or a pharmaceutically acceptable salt thereof, wherein the component present on the immune cell is CD137, and the CD137-binding bicyclic peptide ligand is C i IEEGQYC ii FADPY[Nle]C iii (Sequence No. 1);C i [tBuAla]PE[D-Ala]PYC ii FADPY[Nle]C iii (Sequence No. 3);C i IEEGQYC ii F[D-Ala]DPY[Nle]C iii (Sequence No. 4);C i [tBuAla]PK[D-Ala]PYC ii FADPY[Nle]C iii (Sequence No. 5);C i [tBuAla]PE[D-Lys]PYC ii FADPY[Nle]C iii (Sequence No. 6);C i [tBuAla]P[K(PYA)][D-Ala]PYC ii FADPY[Nle]C iii (Sequence No. 7);C i [tBuAla]PE[D-Lys(PYA)]PYC ii FADPY[Nle]C iii (Sequence No. 8);C i IEE[D-Lys(PYA)]QYC ii FADPY(Nle)C iii (sequence number 9); and [dC i ][dI][dE][dE][K(PYA)][dQ][dY][dC ii ][dF][dA][dD][dP][dY][dNle][dC iii ] comprising an amino acid sequence selected from (SEQ No. 10), or a pharmaceutically acceptable salt thereof, wherein the component present on cancer cells is Nectin-4, and the Nectin-4 binding bicyclic peptide ligand is C i P[1Nal][dD]C ii M[HArg]DWSTP[HyP]WC iii (Sequence No. 15; hereinafter referred to herein as BCY8116);C i P[1Nal][dD]C ii M[HArg]D[dW]STP[HyP][dW]C iii (Sequence No. 16; hereinafter referred to herein as BCY11415);C i P[1Nal][dK](Sar 10 -(B-Ala))C ii M[HArg]DWSTP[HyP]WC iii (Sequence No. 17); and C i PFGC ii M[HArg]DWSTP[HyP]WC iii It comprises an amino acid sequence selected from (SEQ No. 18; hereinafter referred to herein as BCY11414) or a pharmaceutically acceptable salt thereof, wherein C i , C ii and C iii α represents the first, second, and third cysteine ​​residues, respectively; Nle represents norleucine; tBuAla represents t-butyl-alanine; PYA represents 4-pentinoic acid; HyP represents hydroxyproline; dD represents D-configured aspartic acid; HArg represents homoarginine; and Sar 10 A bicyclic peptide complex or a pharmaceutically acceptable salt thereof, comprising: a polypeptide comprising 10 sarcosine units, wherein B-Ala represents beta-alanine and 1Nal represents 1-naphthylalanine, wherein each of the peptide ligands comprises at least three cysteine ​​residues separated by at least two loop sequences; and a molecular scaffold that forms covalent bonds with the cysteine ​​residues of the polypeptide such that at least two polypeptide loops are formed on the molecular scaffold. Claim 2 A bicyclic peptide complex or a pharmaceutically acceptable salt thereof, wherein the immune cell is selected from leukocytes; lymphocytes; CD8 or CD4; CD8; dendritic cells; follicular dendritic cells; and granulocytes. Claim 3 In paragraph 2, a bicyclic peptide complex or a pharmaceutically acceptable salt thereof in which the lymphocytes are T lymphocytes, T cells, B cells, or natural killer cells. Claim 4 In claim 1, the CD137-binding bicyclic peptide ligand comprises N- and C-terminal modifications, and Ac-A-(SEQ No. 1)-Dap (hereinafter referred to herein as BCY7732); Ac-A-(SEQ No. 1)-Dap(PYA) (hereinafter referred to herein as BCY7741); Ac-(SEQ No. 3)-Dap (hereinafter referred to herein as BCY9172); Ac-(SEQ No. 3)-Dap(PYA) (hereinafter referred to herein as BCY11014); Ac-A-(SEQ No. 4)-Dap (hereinafter referred to herein as BCY8045); Ac-(SEQ No. 5)-A (hereinafter referred to herein as BCY8919); Ac-(SEQ No. 6)-A (hereinafter referred to herein as BCY8920); Ac-(SEQ No. 7)-A (hereinafter referred to herein as BCY8927); Ac-(SEQ No. 8)-A (hereinafter referred to herein as BCY8928); Ac-A-(SEQ No. 9)-A (hereinafter referred to herein as BCY7744); or Ac-[dA]-(SEQ No. 10)-[dA]-NH2 (hereinafter referred to herein as BCY11506); or a pharmaceutically acceptable salt thereof comprising a bicyclic peptide complex or a pharmaceutically acceptable salt thereof, wherein Ac represents an acetyl group, Dap represents diaminopropionic acid, and PYA represents 4-pentinoic acid. Claim 5 In claim 4, a CD137-bound bicyclic peptide ligand comprises Ac-(SEQ No. 8)-A (hereinafter referred to herein as BCY8928), wherein Ac represents an acetyl group, a bicyclic peptide complex or a pharmaceutically acceptable salt thereof. Claim 6 In claim 1, a bicyclic peptide complex or a pharmaceutically acceptable salt thereof, wherein the cancer cells are selected from HT1080, SC-OV-3, PC3, H1376, NCI-H292, LnCap, MC38, and RKO tumor cells. Claim 7 In claim 1, the nectin-4 binding bicyclic peptide ligand optionally comprises an N-terminal modification, SEQ ID NO. 15 (hereinafter referred to herein as BCY8116); [PYA]-[B-Ala]-[Sar 10 ]-(Sequence No. 15) (hereinafter referred to herein as BCY8846);Sequence No. 16 (hereinafter referred to herein as BCY11415);[PYA]-[B-Ala]-[Sar 10 ]-(SEQ No. 16) (hereinafter referred to herein as BCY11942); Ac-(SEQ No. 17) (hereinafter referred to herein as BCY8831); or SEQ No. 18 (hereinafter referred to herein as BCY11414); or comprising a pharmaceutically acceptable salt thereof, wherein PYA represents 4-pentinoic acid, B-Ala represents beta-alanine, and Sar 10 A bicyclic peptide complex or a pharmaceutically acceptable salt thereof representing these 10 sarcosine units. Claim 8 A bicyclic peptide complex or a pharmaceutically acceptable salt thereof, wherein the nectin-4 binding bicyclic peptide ligand comprises SEQ ID NO. 15 (hereinafter referred to herein as BCY8116). Claim 9 The CD137 / Nectin-4 complex of claim 1 or 7, comprising a first peptide ligand comprising a CD137-binding bicyclic peptide ligand attached to a TATA scaffold, and a second peptide ligand comprising a Nectin-4-binding bicyclic peptide ligand attached to a TATA scaffold, wherein the complex comprises a bicyclic peptide complex or a pharmaceutically acceptable salt thereof comprising: [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO. 15) (BCY8846) and Ac-A-(SEQ ID NO. 1)-Dap (BCY7732): where the N-terminal PYA of BCY8846 is -PEG to the C-terminal Dap of BCY7732 12 - Linked via linker; · [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID 16) (BCY11942) and Ac-A-(SEQ ID 1)-Dap (BCY7732): where the N-terminal PYA of BCY11942 is -PEG to the C-terminal Dap of BCY7732 12 - Linked via linker; · [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO. 15) (BCY8846) and Ac-A-(SEQ ID NO. 4)-Dap (BCY8045): where the N-terminal PYA of BCY8846 is -PEG to the C-terminal Dap of BCY8045 12 - Connected via linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminus of Dap(PYA) of BCY7741 via -PEG 10 - Connected via linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminus of Dap(PYA) of BCY7741 via -PEG 23 - Connected via linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminal Dap(PYA) of BCY7741 as -B-Ala-Sar 20 - Connected via linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminal Dap(PYA) of BCY7741 as -B-Ala-Sar 10 -PEG 10 - Connected via linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminus of Dap(PYA) of BCY7741, -B-Ala-Sar5-PEG 15 - Connected via a linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminal Dap(PYA) of BCY7741 via a -B-Ala-Sar5-PEG5- linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminal Dap(PYA) of BCY7741 via -PEG 15 Connected via -Sar5- linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminus of Dap(PYA) of BCY7741 via -PEG 10 -Sar 10 - Connected via linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminus of Dap(PYA) of BCY7741 -PEG5-Sar 15 Connected via a linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminal Dap(PYA) of BCY7741 via a -PEG5-Sar5- linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap(PYA) (BCY7741): where the N-terminus of BCY8116 is connected to the C-terminal Dap(PYA) of BCY7741 via a -PEG5- linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 1)-Dap (BCY7732): where the N-terminus of BCY8116 is connected to the C-terminal Dap of BCY7732 via a -PEG 24 - Linked via linker; · [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO. 15) (BCY8846) and Ac-(SEQ ID NO. 3)-Dap (BCY9172): where the N-terminal PYA of BCY8846 and the C-terminal Dap of BCY9172 are -PEG 12 - Linked via linker; · [PYA]-[B-Ala]-[Sar 10 ]-(SEQ No. 15) (BCY8846) and Ac-(SEQ No. 5)-A (BCY8919): where the N-terminal PYA of BCY8846 and the Lys3 of BCY8919 are -PEG 12 - Linked via linker; · [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID 15) (BCY8846) and Ac-(SEQ ID 6)-A (BCY8920): where the N-terminal PYA of BCY8846 and dLys4 of BCY8920 are -PEG 12 - Connected via a linker; · SEQ ID NO. 15 (BCY8116) and Ac-(SEQ ID NO. 7)-A (BCY8927): where the N-terminus of BCY8116 and Lys(PYA)3 of BCY8927 are connected via a -PEG5 linker; · SEQ ID NO. 15 (BCY8116) and Ac-(SEQ ID NO. 8)-A (BCY8928): where the N-terminus of BCY8116 and dLys(PYA)4 of BCY8928 are connected via a -PEG5 linker; · SEQ ID NO. 15 (BCY8116) and Ac-(SEQ ID NO. 3)-Dap(PYA) (BCY11014): where the N-terminus of BCY8116 and the C-terminal Dap(PYA) of BCY11014 are connected via a -PEG5 linker; · [PYA]-[B-Ala]-[Sar 10 ]-(SEQ ID NO. 15) (BCY8846) and Ac-(SEQ ID NO. 3)-Dap (BCY9172): where the N-terminal PYA of BCY8846 and the C-terminal Dap of BCY9172 are connected via a -CH2- linker; · Ac-(SEQ ID NO. 17) (BCY8831) and Ac-(SEQ ID NO. 3)-Dap(PYA) (BCY11014): where the dLys(Sar of BCY8831 10 The C-terminal Dap(PYA) of )-(B-Ala)4 and BCY11014 is -PEG 12 - Linked via linker; · Ac-(SEQ No. 17) (BCY8831) and Ac-(SEQ No. 3)-Dap(PYA) (BCY11014): where, dLys(Sar of BCY8831 10 )-(B-Ala)4 and the C-terminal Dap(PYA) of BCY11014 are linked via a -PEG5- linker; · SEQ ID NO. 15 (BCY8116) and Ac-(SEQ ID NO. 7)-A (BCY8927): where the N-terminus of BCY8116 and the Lys(PYA)3 of BCY8927 are linked via a -CH2- linker; · SEQ ID NO. 15 (BCY8116) and Ac-(SEQ ID NO. 8)-A (BCY8928): where the N-terminus of BCY8116 and the dLys(PYA)4 of BCY8928 are linked via a -CH2- linker; · SEQ ID NO. 15 (BCY8116) and Ac-(SEQ ID NO. 3)-Dap(PYA) (BCY11014): where the N-terminus of BCY8116 and The C-terminal Dap(PYA) of BCY11014 is connected via a -CH2- linker; · SEQ ID NO. 15 (BCY8116) and Ac-A-(SEQ ID NO. 9)-A (BCY7744): where the N-terminus of BCY8116 and dLys(PYA)4 of BCY7744 are connected via a -PEG5- linker; · SEQ ID NO. 15 (BCY8116) and Ac-[dA]-(SEQ ID NO. 10)-[dA]-NH2(BCY11506): where the N-terminus of BCY8116 and Lys(PYA)4 of BCY11506 are connected via a -PEG5- linker; · SEQ ID NO. 18 (BCY11414) and Ac-A-(SEQ ID NO. 9)-A (BCY7744): where BCY11414's The N-terminus and dLys(PYA)4 of BCY7744 are linked via a -PEG5- linker; · SEQ ID NO. 18 (BCY11414) and Ac-(SEQ ID NO. 8)-A (BCY8928): where the N-terminus of BCY11414 and dLys(PYA)4 of BCY8928 are linked via a -PEG5- linker; · SEQ ID NO. 16 (BCY11415) and Ac-(SEQ ID NO. 8)-A (BCY8928): where the N-terminus of BCY11415 and dLys(PYA)4 of BCY8928 are linked via a -PEG5- linker; or a pharmaceutically acceptable salt thereof. Claim 10 In claim 1 or 7, a bicyclic peptide complex or a pharmaceutically acceptable salt thereof, which is a CD137 / nectin-4 complex selected from the following: BCY8854 ;BCY9350 ;BCY9351 ;BCY9399 ;BCY9400 ;BCY9401 ;BCY9403 ;BCY9405 ;BCY9406 ;BCY9407 ;BCY9408 ;BCY9409 ;BCY9410 ;BCY9411 ;BCY9759 ;BCY10000 ;BCY10567 ;BCY10569 ;BCY10571 ;BCY10572 ;BCY10573 ;BCY10578 ;BCY10917 ;BCY11020 ;BCY11373 ;BCY11374 ;BCY11375 ;BCY11616 ;BCY11617 ;BCY11857 ;BCY11858 ;BCY11859 ; or a pharmaceutically acceptable salt thereof. Claim 11 In paragraph 1, the linker is -CH2-, -PEG5-, -PEG 10 -, -PEG 12 -, -PEG 23 -, -PEG 24 -, -PEG 15 -Sar5-, -PEG 10 -Sar 10 -, -PEG5-Sar 15 -, -PEG5-Sar5-, -B-Ala-Sar 20 -, -B-Ala-Sar 10 -PEG 10 -, -B-Ala-Sar5-PEG 15 A bicyclic peptide complex or a pharmaceutically acceptable salt thereof, selected from -, and -B-Ala-Sar5-PEG5-. Claim 12 A bicyclic peptide complex or a pharmaceutically acceptable salt thereof, wherein the molecular scaffold is selected from 1,1',1"-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one (1,1',1''-(1,3,5-triazinane-1,3,5-triyl)triprop-2-en-1-one, TATA). Claim 13 A bicyclic peptide complex or a pharmaceutically acceptable salt thereof, wherein the pharmaceutically acceptable salt is selected from a free acid or sodium, potassium, calcium, and ammonium salts. Claim 14 A bicyclic peptide complex or a pharmaceutically acceptable salt thereof, further comprising a cytotoxic agent, a radiochelating agent, and / or a chromophore, as described in claim 1. Claim 15 A pharmaceutical composition for use in preventing, inhibiting, or treating cancer, comprising a pharmaceutically acceptable carrier, a diluent, or a mixture thereof, and as an active ingredient, the bicyclic peptide complex of claim 1 or a pharmaceutically acceptable salt thereof. Claim 16 A medicine for use in preventing, inhibiting, or treating cancer, comprising as an active ingredient the bicyclic peptide complex of claim 1 or a pharmaceutically acceptable salt thereof. Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete

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